Multi-chain chimeric polypeptides and uses thereof

ABSTRACT

Provided herein are multi-chain chimeric polypeptides that include: 
     (a) a first chimeric polypeptide including a first target-binding domain, a soluble tissue factor domain, and a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide including a second domain of a pair of affinity domains and a second target-binding domain, where the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains. Also provided here are methods of using these multi-chain chimeric polypeptides and nucleic acids encoding these multi-chain chimeric polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to: U.S. Patent Application No.62/724,969, filed Aug. 30, 2018; U.S. Patent Application No. 62/817,230,filed Mar. 12, 2019; U.S. Patent Application Ser. No. 62/725,043, filedAug. 30, 2018; U.S. Patent Application Ser. No. 62/725,010, filed Aug.30, 2018; U.S. Patent Application Ser. No. 62/749,007, filed Oct. 22,2018; U.S. Patent Application Ser. No. 62/746,832, filed Oct. 17, 2018;U.S. Patent Application Ser. No. 62/749,506, filed Oct. 23, 2018; U.S.Patent Application Ser. No. 62/817,241, filed Mar. 12, 2019; U.S. PatentApplication Ser. No. 62/816,683, filed Mar. 11, 2019; and U.S. PatentApplication Ser. No. 62/881,088, filed Jul. 31, 2019, each of which isherein incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to the field of biotechnology, and morespecifically, to antigen-binding molecules.

BACKGROUND

Tissue factor (TF), a 263 amino acid integral membrane glycoprotein witha molecular weight of ˜46 kDa and the trigger protein of the extrinsicblood coagulation pathway, is the primary initiator of coagulation invivo. Tissue factor, normally not in contact with circulating blood,initiates the coagulation cascade upon exposure to the circulatingcoagulation serine protease factors. Vascular damage exposessub-endothelial cells expressing tissue factor, resulting in theformation of a calcium-dependent, high-affinity complex withpre-existing plasma factor VIIa (FVIIa). Binding of the serine proteaseFVIIa to tissue factor promotes rapid cleavage of FX to FXa and FIX toFIXa. The proteolytic activity of the resulting FXa and an activemembrane surface then inefficiently converts a small amount ofprothrombin to thrombin. The thrombin generated by FXa initiatesplatelet activation and activates minute amounts of the pro-cofactorsfactor V (FV) and factor VIII (FVIII) to become active cofactors, factorVa (FVa) and factor VIIIa (FVIIIa). FIXa complexes with FVIIIa on theplatelet surface forming the intrinsic tenase complex, which results inrapid generation of FXa. FXa complexes with FVa to form thepro-thrombinase complex on the activated platelet surface which resultsin rapid cleavage of prothrombin to thrombin.

In addition to the tissue factor-FVIIa complex, a recent study showedthat the tissue factor-FVIIa-FXa complex can activate FVIII, which wouldprovide additional levels of FVIIIa during the initiation phase. Theextrinsic pathway is paramount in initiating coagulation via theactivation of limited amounts of thrombin, whereas the intrinsic pathwaymaintains coagulation by dramatic amplification of the initial signal.

Much of the tissue factor expressed on a cell surface is “encrypted,”which must be “decrypted” for full participation in coagulation. Themechanism of “decryption” of cell-surface tissue factor is still unclearat this time, however, exposure of anionic phospholipids plays a majorrole in this process. Healthy cells actively sequester anionicphospholipids such as phosphatidyl serine (PS) to the inner leaflet ofthe plasma membrane. Following cellular damage, activation, or increasedlevels of cytosolic Ca²⁺, this bilayer asymmetry is lost, resulting inincreased PS exposure on the outer leaflet, which increases the specificactivity of cell-surface tissue factor-FVIIa complexes. PS exposure isknown to decrease the apparent Km for activation of FIX and FX by tissuefactor-FVIIa complexes, but additional mechanisms could includeconformational rearrangement of tissue factor or tissue factor-FVIIa andsubsequent exposure of substrate binding sites.

SUMMARY

The present invention is based on the discovery that soluble tissuefactor can be used as a scaffold for chimeric polypeptides including anantigen-binding domain. Based on this discovery provided herein aremulti-chain chimeric polypeptides that include: (a) a first chimericpolypeptide including: (i) a first target-binding domain; (ii) a solubletissue factor domain; and (iii) a first domain of a pair of affinitydomains; and (b) a second chimeric polypeptide including: (i) a seconddomain of a pair of affinity domains; and (ii) a second target-bindingdomain, where the first chimeric polypeptide and the second chimericpolypeptide associate through the binding of the first domain and thesecond domain of the pair of affinity domains. Also provided herein arecompositions that include any of the multi-chain chimeric polypeptidesdescribed herein, nucleic acids that encode any of the multi-chainchimeric polypeptides described herein, and cells that include any ofthe nucleic acids that encode any of the multi-chain chimericpolypeptides described herein. Also provided herein are methods ofstimulating an immune cell and methods of treating a subject in needthereof that include the use of any of the multi-chain chimericpolypeptides described herein. Also provided herein are methods ofproducing any of the multi-chain chimeric polypeptides described herein.

Accordingly, provided herein is a multi-chain chimeric polypeptidecomprising:

(a) a first chimeric polypeptide comprising:

-   -   (i) a first target-binding domain;    -   (ii) a soluble tissue factor domain; and    -   (iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

-   -   (i) a second domain of a pair of affinity domains; and    -   (ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimericpolypeptide associate through the binding of the first domain and thesecond domain of the pair of affinity domains. In some embodiments, thefirst target-binding domain and the soluble tissue factor domaindirectly abut each other in the first chimeric polypeptide. In someembodiments, the first chimeric polypeptide further comprises a linkersequence between the first target-binding domain and the soluble tissuefactor domain in the first chimeric polypeptide. In some embodiments,the soluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments, the first chimeric polypeptide furthercomprises a linker sequence between the soluble tissue factor domain andthe first domain of the pair of affinity domains in the first chimericpolypeptide. In some embodiments, the second domain of the pair ofaffinity domains and the second target-binding domain directly abut eachother in the second chimeric polypeptide. In some embodiments, thesecond chimeric polypeptide further comprises a linker sequence betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide. In someembodiments, the first target-binding domain and the secondtarget-binding domain bind specifically to the same antigen. In someembodiments, the first target-binding domain and the secondtarget-binding domain bind specifically to the same epitope. In someembodiments, the first target-binding domain and the secondtarget-binding domain comprise the same amino acid sequence. In someembodiments, the first target-binding domain and the secondtarget-binding domain bind specifically to different antigens. In someembodiments, one or both of the first target-binding domain and thesecond target-binding domain is an antigen-binding domain. In someembodiments, the first target-binding domain and the secondtarget-binding domain are each antigen-binding domains. In someembodiments, the antigen-binding domain comprises a scFv or a singledomain antibody. In some embodiments, one or both of the firsttarget-binding domain and the second target-binding domain bindspecifically to a target selected from the group consisting of: CD16a,CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R,IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand ofTGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, aligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand fora scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor forIL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, a receptor for CD122, and a receptor for CD28. Insome embodiments, one or both of the first target-binding domain and thesecond target-binding domain is a soluble interleukin, a solublecytokine protein, or a ligand protein. In some embodiments, the solubleinterleukin, soluble cytokine, or ligand protein is selected from thegroup consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. In some embodiments, oneor both of the first target-binding domain and the second target-bindingdomain is a soluble interleukin, a cytokine receptor, or a soluble cellsurface receptor. In some embodiments, the soluble receptor is a solubleTGF-β receptor II (TGF-β RII), a soluble TGF-βRIII, a soluble NKG2D, asoluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, ascMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28. In someembodiments, the first chimeric polypeptide further comprises one ormore additional target-binding domain(s), where at least one of the oneor more additional antigen-binding domain(s) is positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains. In some embodiments, the first chimeric polypeptidefurther comprises a linker sequence between the soluble tissue factordomain and the at least one of the one or more additionalantigen-binding domain(s), and/or a linker sequence between the at leastone of the one or more additional antigen-binding domain(s) and thefirst domain of the pair of affinity domains. In some embodiments, thefirst chimeric polypeptide further comprises one or more additionaltarget-binding domains at the N-terminal and/or C-terminal end of thefirst chimeric polypeptide. In some embodiments, at least one of the oneor more additional target-binding domains directly abuts the firstdomain of the pair of affinity domains in the first chimericpolypeptide. In some embodiments, the first chimeric polypeptide furthercomprises a linker sequence between the at least one of the one or moreadditional target-binding domains and the first domain of the pair ofaffinity domains. In some embodiments, the at least one of the one ormore additional target-binding domains directly abuts the firsttarget-binding domain in the first chimeric polypeptide. In someembodiments, the first chimeric polypeptide further comprises a linkersequence between the at least one of the one or more additionaltarget-binding domains and the first target-binding domain. In someembodiments, at least one of the one or more additional target-bindingdomains is disposed at the N- and/or C-terminus of the first chimericpolypeptide, and at least one of the one or more additionaltarget-binding domains is positioned between the soluble tissue factordomain and the first domain of the pair of affinity domains in the firstchimeric polypeptide. In some embodiments, the at least one additionaltarget-binding domain of the one or more additional target-bindingdomains disposed at the N-terminus directly abuts the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide. In some embodiments, thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide. In some embodiments, the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the C-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide. In some embodiments, thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide. In some embodiments, the atleast one of the one or more additional target-binding domainspositioned between the soluble tissue factor domain and the first domainof the pair of affinity domains, directly abuts the soluble tissuefactor domain and/or the first domain of the pair of affinity domains.In some embodiments, the first chimeric polypeptide further comprises alinker sequence disposed (i) between the soluble tissue factor domainand the at least one of the one or more additional target-bindingdomains positioned between the soluble tissue factor domain and thefirst domain of the pair of affinity domains, and/or (ii) between thefirst domain of the pair of affinity domains and the at least one of theone or more additional target-binding domains positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains. In some embodiments, the second chimeric polypeptidefurther comprises one or more additional target-binding domains at theN-terminal end or the C-terminal end of the second chimeric polypeptide.In some embodiments, at least one of the one or more additionaltarget-binding domains directly abuts the second domain of the pair ofaffinity domains in the second chimeric polypeptide. In someembodiments, the second chimeric polypeptide further comprises a linkersequence between at least one of the one or more additionaltarget-binding domains and the second domain of the pair of affinitydomains in the second chimeric polypeptide. In some embodiments, atleast one of the one or more additional target-binding domains directlyabuts the second target-binding domain in the second chimericpolypeptide. In some embodiments, the second chimeric polypeptidefurther comprises a linker sequence between at least one of the one ormore additional target-binding domains and the second target-bindingdomain in the second chimeric polypeptide. In some embodiments, two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same antigen. In some embodiments, two or more ofthe first target-binding domain, the second target-binding domain, andthe one or more additional target-binding domains bind specifically tothe same epitope. In some embodiments, two or more of the firsttarget-binding domain, the second target-binding domain, and the one ormore additional target-binding domains comprise the same amino acidsequence. In some embodiments, the first target-binding domain, thesecond target-binding domain, and the one or more additionaltarget-binding domains each bind specifically to the same antigen. Insome embodiments, the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains each bind specifically to the same epitope. In some embodiments,the first target-binding domain, the second target-binding domain, andthe one or more additional target-binding domains each comprise the sameamino acid sequence. In some embodiments, the first target-bindingdomain, the second target-binding domain, and the one or more additionaltarget-binding domains bind specifically to different antigens. In someembodiments, one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more target-binding domains is anantigen-binding domain. In some embodiments, the first target-bindingdomain, the second target-binding domain, and the one or more additionaltarget-binding domains are each an antigen-binding domain. In someembodiments, antigen-binding domain comprises a scFv. In someembodiments, one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more target-binding domains bindspecifically to a target selected from the group consisting of: CD16a,CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R,IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand ofTGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, aligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand fora scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor forIL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, a receptor for CD122, and a receptor for CD3, and areceptor for CD28. In some embodiments, one or more of the firsttarget-binding domain, the second target-binding domain, and the one ormore additional target-binding domains is a soluble interleukin, asoluble cytokine protein, or a ligand protein. In some embodiments, thesoluble interleukin, soluble cytokine, or ligand protein is selectedfrom the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10,IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. In someembodiments, one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains is a soluble interleukin or cytokine receptor. In someembodiments, the soluble receptor is a soluble TGF-β receptor II (TGF-βRII), a soluble TGF-βRIII, a soluble NKG2D, a soluble NKp30, a solubleNKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR,a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. Insome embodiments, the first chimeric polypeptide further comprises apeptide tag at the N-terminal end or the C-terminal end of the firstchimeric polypeptide. In some embodiments, the second chimericpolypeptide further comprises a peptide tag at the N-terminal end or theC-terminal end of the second chimeric polypeptide. In some embodiments,the soluble tissue factor domain is a soluble human tissue factordomain. In some embodiments, the soluble human tissue factor domaincomprises a sequence that is at least 80% identical to SEQ ID NO: 1. Insome embodiments, the soluble human tissue factor domain comprises asequence that is at least 90% identical to SEQ ID NO: 1. In someembodiments, the soluble human tissue factor domain comprises a sequencethat is at least 95% identical to SEQ ID NO: 1. In some embodiments, thesoluble human tissue factor domain does not comprise one or more of: alysine at an amino acid position that corresponds to amino acid position20 of mature wildtype human tissue factor protein; an isoleucine at anamino acid position that corresponds to amino acid position 22 of maturewildtype human tissue factor protein; a tryptophan at an amino acidposition that corresponds to amino acid position 45 of mature wildtypehuman tissue factor protein; an aspartic acid at an amino acid positionthat corresponds to amino acid position 58 of mature wildtype humantissue factor protein; a tyrosine at an amino acid position thatcorresponds to amino acid position 94 of mature wildtype human tissuefactor protein; an arginine at an amino acid position that correspondsto amino acid position 135 of mature wildtype human tissue factorprotein; and a phenylalanine at an amino acid position that correspondsto amino acid position 140 of mature wildtype human tissue factorprotein. In some embodiments, the soluble human tissue factor domaindoes not comprise any of: a lysine at an amino acid position thatcorresponds to amino acid position 20 of mature wildtype human tissuefactor protein; an isoleucine at an amino acid position that correspondsto amino acid position 22 of mature wildtype human tissue factorprotein; a tryptophan at an amino acid position that corresponds toamino acid position 45 of mature wildtype human tissue factor protein;an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein; atyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein; an arginineat an amino acid position that corresponds to amino acid position 135 ofmature wildtype human tissue factor protein; and a phenylalanine at anamino acid position that corresponds to amino acid position 140 ofmature wildtype human tissue factor protein. In some embodiments, thesoluble tissue factor domain is not capable of binding to Factor VIIa.In some embodiments, the soluble tissue factor domain does not convertinactive Factor X into Factor Xa. In some embodiments, the multi-chainchimeric polypeptide does not stimulate blood coagulation in a mammal.In some embodiments of any of the single-chain chimeric polypeptidesprovided herein, the human soluble tissue factor domain does notinitiate blood coagulation. In some embodiments of any of thesingle-chain chimeric polypeptides provided herein, the soluble tissuefactor domain comprises or consists of a soluble wildtype human tissuefactor.

In some embodiments, the pair of affinity domains is a sushi domain froman alpha chain of human IL-15 receptor (IL15Rα) and a soluble IL-15. Insome embodiments, the soluble IL15 has a D8N or D8A amino acidsubstitution. In some embodiments, the human IL15Rα is a maturefull-length IL15Rα. In some embodiments, the pair of affinity domains isselected from the group consisting of: barnase and barnstar, a PKA andan AKAP, adapter/docking tag modules based on mutated RNase I fragments,and SNARE modules based on interactions of the proteins syntaxin,synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the firstchimeric polypeptide and/or the second chimeric polypeptide furthercomprises a signal sequence at its N-terminal end.

In some embodiments, the multi-chain chimeric polypeptide comprises acomposition. In some embodiments, the composition is a pharmaceuticalcomposition. In some embodiments, the composition comprises at least onedose of the multi-chain chimeric polypeptide. In some embodiments, a kitcomprises at least one dose of the composition.

In some embodiments, provided herein is a method of stimulating animmune cell, the method comprising: contacting an immune cell with aneffective amount of any of the multi-chain chimeric polypeptides orcompositions described above. In some embodiments, the method comprisescontacting the immune cell in vitro. In some embodiments, the methodcomprises obtaining the immune cell from a subject. In some embodiments,the method comprises obtaining the immune cell from the subject prior tothe contacting step. In some embodiments, the method comprisescontacting the immune cell in vivo. In some embodiments, the methodcomprises selecting the immune cell from the group consisting of: animmature thymocyte, a peripheral blood lymphocyte, a naïve T cell, apluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell,a memory T cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell,a CD8⁺ T cell, a CD4⁺ T cell, a natural killer T cell, a mast cell, amacrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil,and a natural killer cell. In some embodiments, the method comprisesgenetically modifying the immune cell to express a chimeric antigenreceptor or a recombinant T-cell receptor. In some embodiments, themethod comprises introducing into the immune cell a nucleic acidencoding a chimeric antigen-receptor or a recombinant T-cell receptorafter the contacting step. In some embodiments, the method comprisesadministering the immune cell to a subject in need thereof. In someembodiments, the method comprises identifying or diagnosing the subjectas having an age-related disease or condition. In some embodiments, theage-related disease or condition is selected from the group consistingof: Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction. In some embodiments,the method comprises identifying or diagnosing the subject as having acancer. In some embodiments, the cancer is selected from the groupconsisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma. In some embodiments,the method comprises diagnosing or identifying the subject as having aninfectious disease. In some embodiments, the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Also provided herein is a method of inducing or increasing proliferationof an immune cell, the method comprising: contacting an immune cell withan effective amount of any of the multi-chain chimeric polypeptides orcompositions described above. In some embodiments, the method comprisescontacting the immune cell in vitro. In some embodiments, the methodcomprises obtaining the immune cell from a subject. In some embodiments,the method comprises obtaining the immune cell from the subject prior tothe contacting step. In some embodiments, the method comprisescontacting the immune cell in vivo. In some embodiments, the methodcomprises selecting the immune cell from the group consisting of: animmature thymocyte, a peripheral blood lymphocyte, a naïve T cell, apluripotent Th cell precursor, a lymphoid progenitor cell, a Treg cell,a memory T cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell,a CD8⁺ T cell, a CD4⁺ T cell, a natural killer T cell, a mast cell, amacrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil,and a natural killer cell. In some embodiments, the method comprisesmodifying the immune cell to express a chimeric antigen receptor or arecombinant T-cell receptor. In some embodiments, the method furthercomprises introducing into the immune cell a nucleic acid encoding achimeric antigen-receptor or a recombinant T-cell receptor after thecontacting step. In some embodiments, the method further comprisesadministering the immune cell to a subject in need thereof. In someembodiments, the method comprises identifying or diagnosing the subjectas having an age-related disease or condition. In some embodiments, theage-related disease or condition is selected from the group consistingof: Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction. In some embodiments,the method comprises identifying or diagnosing the subject as having acancer. In some embodiments, the cancer is selected from the groupconsisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma. In some embodiments,the method comprises diagnosing or identifying the subject as having aninfectious disease. In some embodiments, the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Also provided herein is a method of inducing differentiation of animmune cell into a memory or memory-like immune cell, the methodcomprising: contacting an immune cell with an effective amount of any ofthe multi-chain chimeric polypeptides or compositions discussed above.In some embodiments, the method comprises contacting the immune cell invitro. In some embodiments, the method comprises obtaining the immunecell from a subject. In some embodiments, the method further comprisesobtaining the immune cell from the subject prior to the contacting step.In some embodiments, the method comprises contacting the immune cell invivo. In some embodiments, the immune cell is selected from the groupconsisting of: an immature thymocyte, a peripheral blood lymphocyte, anaïve T cell, a pluripotent Th cell precursor, a lymphoid progenitorcell, a Treg cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, aTh1 cell, a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating Tcell, a CD8+ T cell, a CD4+ T cell, a natural killer T cell, a mastcell, a macrophage, a neutrophil, a dendritic cell, a basophil, aneosinophil, and a natural killer cell. In some embodiments, the immunecell has previously been genetically modified to express a chimericantigen receptor or a recombinant T-cell receptor. In some embodiments,the method further comprises introducing into the immune cell a nucleicacid encoding a chimeric antigen-receptor or a recombinant T-cellreceptor after the contacting step. In some embodiments, the methodfurther comprises administering the immune cell to a subject in needthereof. In some embodiments, the method comprises identifying ordiagnosing the subject as having an age-related disease or condition. Insome embodiments, the age-related disease or condition is selected fromthe group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis,fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonaryfibrosis, inflammatory bowel disease, intervertebral disc degeneration,macular degeneration, osteoarthritis, type 2 diabetes mellitus, adiposeatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathicpulmonary fibrosis, kidney transplant failure, liver fibrosis, loss ofbone mass, myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction. In some embodiments,the method comprises identifying or diagnosing the subject as having acancer. In some embodiments, the cancer is selected from the groupconsisting of: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma. In some embodiments,the method comprises diagnosing or identifying the subject as having aninfectious disease. In some embodiments, the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Also provided herein is a method of killing a cancer cell, an infectedcell, or a senescent cell in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of any of the multi-chain chimeric polypeptides or compositionsdiscussed above. In some embodiments, the method comprises identifyingor diagnosing the subject as having a cancer. In some embodiments, thecancer is selected from the group consisting of: solid tumor,hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma,melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cellneoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin'slymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML), acutelymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneousT-cell lymphoma, retinoblastoma, stomach cancer, urothelial carcinoma,lung cancer, renal cell carcinoma, gastric and esophageal cancer,pancreatic cancer, prostate cancer, breast cancer, colorectal cancer,ovarian cancer, non-small cell lung carcinoma, squamous cell head andneck carcinoma, endometrial cancer, cervical cancer, liver cancer, andhepatocellular carcinoma. In some embodiments, the method comprisesidentifying or diagnosing the subject as having an aging-related diseaseor condition. In some embodiments, the aging-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction. In some embodiments, themethod comprises administering to the subject a therapeuticallyeffective amount of any of the multi-chain chimeric polypeptides orcompositions discussed above. In some embodiments, the method comprisesidentifying or diagnosing the subject as having a cancer. In someembodiments, the cancer is selected from the group consisting of: solidtumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma,neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma,B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cellnon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia(CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML),acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS),cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelialcarcinoma, lung cancer, renal cell carcinoma, gastric and esophagealcancer, pancreatic cancer, prostate cancer, breast cancer, colorectalcancer, ovarian cancer, non-small cell lung carcinoma, squamous cellhead and neck carcinoma, endometrial cancer, cervical cancer, livercancer, and hepatocellular carcinoma. In some embodiments, the methodcomprises identifying or diagnosing the subject as having anaging-related disease or condition. In some embodiments, theaging-related disease or condition is selected from the group consistingof: Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis inpancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis,inflammatory bowel disease, intervertebral disc degeneration, maculardegeneration, osteoarthritis, type 2 diabetes mellitus, adipose atrophy,lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonaryfibrosis, kidney transplant failure, liver fibrosis, loss of bone mass,myocardial infarction, sarcopenia, wound healing, alopecia,cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease,age-associated loss of lung tissue elasticity, macular degeneration,cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis,amyotrophic lateral sclerosis, Huntington's disease, spinocerebellarataxia, multiple sclerosis, and renal dysfunction. In some embodiments,the method comprises diagnosing or identifying the subject as having aninfectious disease. In some embodiments, the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Also provided herein is a nucleic acid encoding any of the multi-chainchimeric polypeptides discussed above. Some embodiments comprise vectorcontaining any of the nucleic acids discussed above. In someembodiments, the vector is an expression vector. Some embodimentscomprise a cell containing any of the nucleic acids discussed above.

Also provided herein is a method of producing a multi-chain chimericpolypeptide, the method comprising: culturing the cell discussed abovein a culture medium under conditions sufficient to result in theproduction of the multi-chain chimeric polypeptide; and recovering themulti-chain chimeric polypeptide from the cell and/or the culturemedium. In some embodiments, the method comprises producing amulti-chain chimeric polypeptide by the methods discussed above.

In some embodiments, the mutant soluble human tissue factor domaincomprises a sequence that is at least 80% identical to SEQ ID NO: 3. Insome embodiments, the soluble human tissue factor domain comprises asequence that is at least 90% identical to SEQ ID NO: 3. In someembodiments, the soluble human tissue factor domain comprises a sequencethat is at least 95% identical to SEQ ID NO: 3. In some embodiments, themulti-chain chimeric polypeptide of claim 140, wherein the soluble humantissue factor domain comprises a sequence that is 100% identical to SEQID NO: 3. In some embodiments, the soluble human tissue factor domaincomprises a sequence that is at least 80% identical to SEQ ID NO: 4. Insome embodiments, the soluble human tissue factor domain comprises asequence that is at least 90% identical to SEQ ID NO: 4. In someembodiments, the soluble human tissue factor domain comprises a sequencethat is at least 95% identical to SEQ ID NO:4. In some embodiments, thesoluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 4.

As used herein, the term “chimeric” refers to a polypeptide thatincludes amino acid sequences (e.g., domains) originally derived fromtwo different sources (e.g., two different naturally-occurring proteins,e.g., from the same or different species). For example, a chimericpolypeptide can include domains from at least two different naturallyoccurring human proteins. In some examples, a chimeric polypeptide caninclude a domain that is a synthetic sequence (e.g., an scFv) and adomain that is derived from a naturally-occurring protein (e.g., anaturally-occurring human protein). In some embodiments, a chimericpolypeptide can include at least two different domains that aresynthetic sequences (e.g., two different scFvs).

An “antigen-binding domain” is one or more protein domain(s) (e.g.,formed from amino acids from a single polypeptide or formed from aminoacids from two or more polypeptides (e.g., the same or differentpolypeptides) that is capable of specifically binding to one or moredifferent antigen(s). In some examples, an antigen-binding domain canbind to an antigen or epitope with specificity and affinity similar tothat of naturally-occurring antibodies. In some embodiments, theantigen-binding domain can be an antibody or a fragment thereof. In someembodiments, an antigen-binding domain can include an alternativescaffold. Non-limiting examples of antigen-binding domains are describedherein. Additional examples of antigen-binding domains are known in theart.

A “soluble tissue factor domain” refers to a polypeptide having at least70% identity (e.g., at least 75% identity, at least 80% identity, atleast 85% identity, at least 90% identity, at least 95% identity, atleast 99% identity, or 100% identical) to a segment of a wildtypemammalian tissue factor protein (e.g., a wildtype human tissue factorprotein) that lacks the transmembrane domain and the intracellulardomain. Non-limiting examples of soluble tissue factor domains aredescribed herein.

The term “soluble interleukin protein” is used herein to refer to amature and secreted interleukin protein or a biologically activefragment thereof. In some examples, a soluble interleukin protein caninclude a sequence that is at least 70% identical, at least 75%identical, at least 80% identical, at least 85% identical, at least 90%identical, at least 95% identical, at least 99% identical, or 100%identical to a wildtype mature and secreted mammalian interleukinprotein (e.g., a wildtype human interleukin protein) and retains itsbiological activity. Non-limiting examples of soluble interleukinproteins are described herein.

The term “soluble cytokine protein” is used herein to refer to a matureand secreted cytokine protein or a biologically active fragment thereof.In some examples, a soluble cytokine protein can include a sequence thatis at least 70% identical, at least 75% identical, at least 80%identical, at least 85% identical, at least 90% identical, at least 95%identical, at least 99% identical, or 100% identical to a wildtypemature and secreted mammalian interleukin protein (e.g., a wildtypehuman interleukin protein) and retains its biological activity.Non-limiting examples of soluble cytokine proteins are described herein.

The term “soluble interleukin receptor” is used herein in the broadestsense to refer to a polypeptide that lacks a transmembrane domain (andoptionally an intracellular domain) that is capable of binding one ormore of its natural ligands (e.g., under physiological conditions, e.g.,in phosphate buffered saline at room temperature). For example, asoluble interleukin receptor can include a sequence that is at least 70%identical (e.g., at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, atleast 99% identical, or 100% identical) to an extracellular domain ofwildtype interleukin receptor and retains its ability to specificallybind to one or more of its natural ligands, but lacks its transmembranedomain (and optionally, further lacks its intracellular domain).Non-limiting examples of soluble interleukin receptors are describedherein.

The term “soluble cytokine receptor” is used herein in the broadestsense to refer to a polypeptide that lacks a transmembrane domain (andoptionally an intracellular domain) that is capable of binding one ormore of its natural ligands (e.g., under physiological conditions, e.g.,in phosphate buffered saline at room temperature). For example, asoluble cytokine receptor can include a sequence that is at least 70%identical (e.g., at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, atleast 99% identical, or 100% identical) to an extracellular domain ofwildtype cytokine receptor and retains its ability to specifically bindto one or more of its natural ligands, but lacks its transmembranedomain (and optionally, further lacks its intracellular domain).Non-limiting examples of soluble cytokine receptors are describedherein.

The term “antibody” is used herein in its broadest sense and includescertain types of immunoglobulin molecules that include one or moreantigen-binding domains that specifically bind to an antigen or epitope.An antibody specifically includes, e.g., intact antibodies (e.g., intactimmunoglobulins), antibody fragments, and multi-specific antibodies. Oneexample of an antigen-binding domain is an antigen-binding domain formedby a VH-VL dimer. Additional examples of an antibody are describedherein. Additional examples of an antibody are known in the art.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between an antigen-binding site and its binding partner(e.g., an antigen or epitope). Unless indicated otherwise, as usedherein, “affinity” refers to intrinsic binding affinity, which reflectsa 1:1 interaction between members of an antigen-binding domain and anantigen or epitope. The affinity of a molecule X for its partner Y canbe represented by the dissociation equilibrium constant (K_(D)). Thekinetic components that contribute to the dissociation equilibriumconstant are described in more detail below. Affinity can be measured bycommon methods known in the art, including those described herein.Affinity can be determined, for example, using surface plasmon resonance(SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g.,FORTEBIO®). Additional methods for determining the affinity for anantigen-binding domain and its corresponding antigen or epitope areknown in the art.

A “multi-chain polypeptide” as used herein to refers to a polypeptidecomprising two or more (e.g., three, four, five, six, seven, eight,nine, or ten) protein chains (e.g., at least a first chimericpolypeptide and a second polypeptide), where the two or more proteinschains associate through non-covalent bonds to form a quaternarystructure. The term “pair of affinity domains” is two different proteindomain(s) that bind specifically to each other with a K_(D) of less thanof less than 1×10⁻⁷ M (e.g., less than 1×10⁻⁸M, less than 1×10⁻⁹M, lessthan 1×10⁻¹⁰ M, or less than 1×10⁻¹¹M). In some examples, a pair ofaffinity domains can be a pair of naturally-occurring proteins. In someembodiments, a pair of affinity domains can be a pair of syntheticproteins. Non-limiting examples of pairs of affinity domains aredescribed herein.

The term “epitope” means a portion of an antigen that specifically bindsto an antigen-binding domain. Epitopes can, e.g., consist ofsurface-accessible amino acid residues and/or sugar side chains and mayhave specific three-dimensional structural characteristics, as well asspecific charge characteristics. Conformational and non-conformationalepitopes are distinguished in that the binding to the former but not thelatter may be lost in the presence of denaturing solvents. An epitopemay comprise amino acid residues that are directly involved in thebinding, and other amino acid residues, which are not directly involvedin the binding. Methods for identifying an epitope to which anantigen-binding domain binds are known in the art.

An “immune effector cell” refers to a cell of the immune system of amammal that is capable, directly or indirectly, of recognizing and/orcausing cytostasis or cell death of a pathogenic cell (e.g., a cancercell) in the mammal. Non-limiting examples of immune effector cellsinclude macrophages, T-lymphocytes (e.g., cytotoxic T-lymphocytes andT-helper cells), natural killer cells, neutrophils, monocytes, andeosinophils. Additional examples of immune effector cells are known inthe art.

The term “treatment” means to ameliorate at least one symptom of adisorder. In some examples, the disorder being treated is cancer and toameliorate at least one symptom of cancer includes reducing aberrantproliferation, gene expression, signaling, translation, and/or secretionof factors. Generally, the methods of treatment include administering atherapeutically effective amount of composition that reduces at leastone symptom of a disorder to a subject who is in need of, or who hasbeen determined to be in need of such treatment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows exemplary diagrams for a multi-chain chimeric polypeptide:(i) a first chimeric polypeptide including a first target-binding domain(A), a soluble tissue factor domain, a first domain of an affinity pairof domains (soluble interleukin IL-15), and an additional target-bindingdomain (B); and (ii) second chimeric polypeptide including a seconddomain of an affinity pair of domains (IL-15 receptor alpha sushidomain), a second target-binding domain (C), and an additionalantigen-binding domain (D). The top cartoon diagram depicts theassociation of the first and the second chimeric polypeptides throughthe pair of affinity domains. The bottom schematic diagrams show theorder of the domains in the first and second chimeric polypeptides.

FIG. 2 shows exemplary diagrams for a multi-chain chimeric polypeptide:(i) a first chimeric polypeptide including a first target-binding domain(A), a soluble tissue factor domain including five amino acidsubstitutions in order to remove binding of the soluble tissue factordomain to FVIIa, a first domain of an affinity pair of domains (solubleinterleukin IL-15 including a D8N or D8A amino acid substitution), andan additional target-binding domain (B); and (ii) second chimericpolypeptide including a second domain of an affinity pair of domains(IL-15 receptor alpha sushi domain), a second target-binding domain (C),and an additional antigen-binding domain (D). The top cartoon diagramdepicts the association of the first and the second chimericpolypeptides through the pair of affinity domains. The bottom schematicdiagrams show the order of the domains in the first and second chimericpolypeptides. In other embodiments of any of the multi-chain chimericpolypeptides described herein the soluble tissue factor domain cancomprise or consists of a soluble wildtype human tissue factor domain(comprising or consisting of a contiguous sequence within wildtype humantissue factor).

FIG. 3 shows a schematic diagram of an exemplary IL-12/IL-15RαSu DNAconstruct.

FIG. 4 shows a schematic diagram of an exemplary IL-18/TF/IL-15 DNAconstruct.

FIG. 5 shows a schematic diagram of the interaction between theexemplary IL-12/IL-15RαSu and IL-18/TF/IL-15 DNA constructs.

FIG. 6 shows a schematic diagram of the interaction between theexemplary IL-12/IL-15RαSu and IL-18/TF/IL-15 fusion proteins resultingin IL-18/TF/IL-15:IL-12/IL-15RαSu complex (18t15-12s).

FIG. 7 shows a chromatograph of 18t15-12s purification elution from ananti-TF antibody affinity column.

FIG. 8 shows an exemplary chromatographic profile of anti-TFAb/SEC-purified 18t15-12s protein following elution on an analyticalsize exclusion column, demonstrating separation of monomericmultiprotein 18t15-12s complexes from protein aggregates.

FIG. 9 shows an example of a 4-12% SDS-PAGE of the 18t15-12s complexfollowing disulfide bond reduction. Lane 1: SeeBlue Plus2 marker; Lane2: an anti-tissue factor antibody affinity column-purified 18t15-12s(0.5 μg); Lane 3: an anti-tissue factor antibody affinitycolumn-purified 18t15-12s (1 μg).

FIG. 10 shows SDS PAGE analysis of deglycosylated and non-deglycosylated18t15-12s. Lane 1: an anti-tissue factor antibody affinitycolumn-purified 18t15-12s (0.5 μg), non-deglycosylated; Lane 2: anti-TFAb-purified 18t15-12s (1 μg), non-deglycosylated; Lane 3: 18t15-12s (1μg), deglycosylated, Lane 4: Mark12 unstained maker.

FIG. 11 shows a sandwich ELISA for the 18t15-12s complex, comprising ananti-human tissue factor capture antibody and a biotinylated anti-humanIL-12 detection antibody (BAF 219).

FIG. 12 shows a sandwich ELISA for the 18t15-12s complex, comprising ananti-human tissue factor capture antibody and a biotinylated anti-humanIL-15 detection antibody (BAM 247).

FIG. 13 shows a sandwich ELISA for the 18t15-12s complex, comprising ananti-human tissue factor capture antibody and a biotinylated anti-humanIL-18 detection antibody (D045-6).

FIG. 14 shows a sandwich ELISA for the 18t15-12s complex, comprising ananti-human tissue factor (143) capture antibody and an anti-human tissuefactor detection antibody.

FIG. 15 shows proliferation of IL-15-dependent 32Dβ cells mediated bythe 18t15-12s complex (open squares) and recombinant IL-15 (blacksquares).

FIG. 16 shows biological activity of IL-18 within the 18t15-12s complex(open squares), where recombinant IL-18 (black squares) and recombinantIL-12 (black circles) serve as positive and negative controls,respectively.

FIG. 17 shows biological activity of IL-12 within the 18t15-12s complex(open squares), where recombinant IL-12 (black circles) and recombinantIL-18 (open squares) serve as positive and negative controls,respectively.

FIGS. 18A and 18B show cell-surface expression of CD25 on NK cellsinduced by the 18t15-12s complex and cell-surface CD69 expression of NKcells induced by the 18t15-12s complex.

FIG. 19 shows a flow cytometry graph of intracellular interferon gammaexpression of NK cells induced by the 18t15-12s complex.

FIG. 20 shows cytotoxicity of 18t15-12s induced human NK cells againstK562 cells.

FIG. 21 shows a schematic diagram of an exemplary IL-12/IL-15RαSu/αCD16DNA construct.

FIG. 22 shows a schematic diagram of an exemplary IL-18/TF/IL-15 DNAconstruct.

FIG. 23 shows a schematic diagram of the interaction between theexemplary IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15 DNA constructs.

FIG. 24 shows a schematic diagram of an exemplary 18t15-12s/αCD16protein complex.

FIG. 25 shows a sandwich ELISA for the 18t15-12s16 complex, comprisingan anti-human tissue factor capture antibody and a biotinylatedanti-human IL-12 or IL-18 detection antibody.

FIG. 26 shows a schematic diagram of an exemplary TGFβRII/IL-15RαSu DNAconstruct.

FIG. 27 shows a schematic diagram of an exemplary IL-21/TF/IL-15construct.

FIG. 28 shows a schematic diagram of the interaction between theexemplary IL-IL-21/TF/IL-15 and TGFβRII/IL-15RαSu constructs.

FIG. 29 shows a schematic diagram of the interaction between theexemplary TGFβRII/IL-15RαSu and IL-21/TF/IL-15 fusion proteins,resulting in an IL-21/TF/IL-15/TGFβRII/IL-15RαSu complex (21t15-TGFRs).

FIG. 30 shows a chromatograph of 21t15-TGFRs purification elution froman anti-TF antibody affinity column.

FIG. 31 shows an exemplary 21t15-TGFRs size exclusion chromatographshowing a main protein peak and a high molecular weight peak

FIG. 32 shows an example of a 4-12% SDS-PAGE of the 21t15-TGFRs complexfollowing disulfide bond reduction. Lane 1: Mark12 unstained marker(numbers on the left side indicate molecular weights in kDa); Lane 2:21t15-TGFRs (0.5 μg); Lane 3: 21t15-TGFRs (1 μg); Lane 4: 21t15-TGFRs,deglycosylated (1 μg), wherein the MW was the expected size of 53 kDaand 39.08 kDa.

FIG. 33 shows a sandwich ELISA for the 21t15-TGFRs complex, comprisingan anti-human tissue factor capture antibody and a biotinylatedanti-human IL-21 detection antibody (13-7218-81, BioLegend).

FIG. 34 shows a sandwich ELISA for the 21t15-TGFRs complex, comprisingan anti-human tissue factor capture antibody and a biotinylatedanti-human IL-15 detection antibody (BAM 247, R&D Systems).

FIG. 35 shows a sandwich ELISA for the 21t15-TGFRs complex, comprisingan anti-human tissue factor capture antibody and a biotinylatedanti-human TGFβRII detection antibody (BAF241, R&D Systems).

FIG. 36 shows a sandwich ELISA for the 21t15-TGFRs complex, comprisingan anti-human tissue factor (143) capture antibody and an anti-humantissue factor detection antibody.

FIG. 37 shows IL-15-dependent proliferation of 32Dβ cells mediated bythe 21t15-TGFRs complex (open squares) compared to IL-15 (blacksquares).

FIG. 38 shows biological activity of the TGFβRII domain within the21t15-TGFRs complex (open squares). TGFβRII/Fc (black squares) served asa positive control.

FIG. 39 A shows a flow cytometry graph of cell-surface CD25 expressionof NK cells induced by the 21t15-TGFRs complex.

FIG. 40 shows a flow cytometry graph of cell-surface CD69 expression ofNK cells induced by the 21t15-TGFRs complex.

FIG. 41 shows a flow cytometry graph of intracellular interferon gammaexpression of NK cells induced by the 21t15-TGFRs complex.

FIG. 42 shows cytotoxicity of 21t15-TGFRs-induced human NK cells againstK562 cells.

FIG. 43 shows a schematic diagram of an exemplary IL-7/IL-15RαSu DNAconstruct.

FIG. 44 shows a schematic diagram of an exemplary IL-21/TF/IL-15 DNAconstruct.

FIG. 45 shows a schematic diagram of the interaction between theexemplary IL-7/IL-15RαSu and IL-21/TF/IL-15 DNA constructs.

FIG. 46 shows a schematic diagram of the interaction between theexemplary IL-7/IL-15RαSu and IL-21/TF/IL-15 fusion proteins resulting inan IL-21/TF/IL-15:IL-7/IL-15RαSu complex (21t15-7s).

FIG. 47 shows the expansion of primary natural killer (NK) cells bystimulation with 21t15-7s+anti-TF IgG1 antibody.

FIG. 48 shows activation of expanded primary NK cells, using CD25 MFIand CD69 MFI as markers of NK cell activation.

FIG. 49 shows cytotoxic activity of expanded NK cells against K562 humantumor cells, wherein NK cells stimulated with 21t15-7s+anti-TF IgG1antibody demonstrate greater specific lysis of K562 cells than NK cellsnot stimulated with 21t15-7s+anti-TF IgG1 antibody.

FIG. 50 shows a schematic diagram of an exemplary IL-21/IL-15RαSu DNAconstruct.

FIG. 51 shows a schematic diagram of an exemplary IL-7/TF/IL-15 DNAconstruct.

FIG. 52 shows a schematic diagram of the interaction between theexemplary IL-21/IL-15RαSu and IL-7/TF/IL-15 DNA constructs.

FIG. 53 shows a schematic diagram of the interaction between theexemplary IL-21/IL-15RαSu and IL-7/TF/IL-15 fusion proteins resulting inan IL-7/TF/IL-15:IL-21/IL-15RαSU complex (7t15-21s).

FIG. 54 shows in diagrammatic form the activation and expansion ofprimary natural killer (NK) cells by stimulation with21t15-TGFRs+anti-TF IgG1 antibody.

FIG. 55 shows size exclusion chromatography (SEC) profiles of anti-TFIgG1 antibody, 7t15-21s and the complex containing equal amounts ofanti-TF IgG1 antibody and 7t15-21s.

FIG. 56 shows the oxygen consumption rate (OCR) in pmoles/min for humanNK cells isolated from blood (2×10⁶ cells/mL) of two different donors.

FIG. 57 shows the extracellular acidification rate (ECAR) in mpH/minutefor human NK cells isolated from blood (2×10⁶ cells/mL) of two differentdonors.

FIG. 58 shows a schematic of the 7t15-16s21 construct.

FIG. 59 shows an additional schematic of the 7t15-16s21 construct.

FIGS. 60A and 60B show binding of 7t15-16s21 to CHO cells expressinghuman CD16b as compared to a control protein.

FIGS. 61A-61C are results from ELISA experiments using antibodiesagainst IL-15, IL-21, and IL-7 in detecting 7t15-16s21.

FIG. 62 shows results of the 32Dβ cell proliferation assay with7t15-16s21 or recombinant IL-15.

FIG. 63 shows the chromatographic profile of 7t15-16s21 proteincontaining cell culture supernatant following binding and elution onanti-TF antibody resin.

FIG. 64 shows the analytical SEC Profile of 7t15-16s21.

FIG. 65 shows a schematic of the TGFRt15-16s21 construct.

FIG. 66 shows an additional schematic of the TGFRt15-16s21 construct.

FIGS. 67A and 67B show binding affinity of TGFRt15-16521 and 7t15-21swith CHO cells expressing human CD16b. FIG. 67A shows binding affinityof TGFRt15-16S21 with CHO cells expressing human CD16b. FIG. 67B showsbinding affinity of 7t15-21s with CHO cells expressing human CD16b.

FIG. 68 shows results of TGFβ1 inhibition by TGFRt15-16s21 and TGFR-Fc.

FIG. 69 shows results of 32Dβ cell proliferation assay withTGFRt15-16s21 or recombinant IL-15.

FIGS. 70A-70C show results of detecting IL-15, IL-21, and TGFβRII inTGFRt15-16s21 with corresponding antibodies using ELISA.

FIG. 71 shows the chromatographic profile of TGFRt15-16s21 proteincontaining cell culture supernatant following binding and elution onanti-TF antibody resin.

FIG. 72 shows results of a reduced SDS-PAGE analysis of TGFRt15-16s21.

FIG. 73 shows a schematic of the 7t15-7s construct.

FIG. 74 shows an additional schematic of the 7t15-7s construct.

FIG. 75 shows the chromatographic profile of 7t15-7s protein containingcell culture supernatant following binding and elution on anti-TFantibody resin.

FIG. 76 shows detection of TF, IL-15 and IL-7 in 7t15-7s using ELISA.

FIG. 77 shows a schematic of the TGFRt15-TGFRs construct.

FIG. 78 shows an additional schematic of the TGFRt15-TGFRs construct.

FIG. 79 shows results of TGFβ1 inhibition by TGFRt15-TGFRs and TGFR-Fc.

FIG. 80 shows results of 32Dβ cell proliferation assay withTGFRt15-TGFRs or recombinant IL-15

FIGS. 81A and 81B show results of detecting IL-15 and TGFβRII inTGFRt15-TGFRs with corresponding antibodies using ELISA.

FIG. 82 is a line graph showing the chromatographic profile ofTGFRt15-TGFRs protein containing cell culture supernatant followingbinding and elution on anti-TF antibody resin.

FIG. 83 shows the analytical SEC profile of TGFRt15-TGFRs.

FIG. 84 shows TGFRt15-TGFRs before and after deglycosylation as analyzedby reduced SDS-PAGE.

FIGS. 85A and 85B show spleen weight and the percentages of immune celltypes in TGFRt15-TGFRs-treated and control-treated mice. FIG. 85A showsspleen weight in mice treated with TGFRt15-TGFRs as compared to PBScontrol. FIG. 85B shows the percentage of CD4⁺ T cells, CD8⁺ T cells,and NK cells in mice treated with TGFRt15-TGFRs as compared to PBScontrol.

FIGS. 86A and 86B show the spleen weight and immunostimulation over 92hours in mice treated with TGFRt15-TGFRs. FIG. 86A shows spleen weightof mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours aftertreatment. FIG. 86B shows the percentages of immune cells in micetreated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours aftertreatment.

FIGS. 87A and 87B show Ki67 and Granzyme B expression in mice treatedwith TGFRt15-TGFRs over time.

FIG. 88 shows enhancement of cytotoxicity of splenocytes byTGFRt15-TGFRs in C57BL/6 Mice.

FIG. 89 shows changes in tumor size in response to PBS treatment,chemotherapy alone, TGFRt15-TGFRs alone, or chemotherapy andTGFRt15-TGFRs combination, in a pancreatic cancer mouse model.

FIG. 90 shows the cytotoxicity of NK cells isolated from mice treatedwith TGFRt15-TGFRs.

FIG. 91 shows a schematic of the 7t15-21s137L (long version) construct.

FIG. 92 shows an additional schematic of the 7t15-21s137L (long version)construct.

FIG. 93 is a line graph showing the chromatographic profile of7t15-21s137L (long version) protein containing cell culture supernatantfollowing binding and elution on anti-TF antibody resin.

FIG. 94 shows the analytical SEC profile of 7t15-21s137L (long version).

FIG. 95 shows binding of 7t15-21s137L (short version) to CD137L (4.1BBL)

FIGS. 96A-96C show detection of IL15, IL21, and IL7 in 7t15-21s137L(short version) with ELISA. FIG. 96A shows detection of IL15 in7t15-21s137L (short version) with ELISA. FIG. 96B shows detection ofIL21 in 7t15-21s137L (short version) with ELISA. FIG. 96C showsdetection of IL7 in 7t15-21s137L (short version) with ELISA.

FIG. 97 shows results from a CTLL-2 cell proliferation assay.

FIG. 98 shows the activity of 7t15-1s137L (short version) in promotingIL21R containing B9 cell proliferation.

FIG. 99 shows a schematic of the 7t15-TGFRs construct.

FIG. 100 shows an additional schematic of the 7t15-TGFRs construct.

FIG. 101 shows results of TGFβ1 inhibition by 7t15-TGFRs and TGFR-Fc.

FIGS. 102A-102C show detection of IL-15, TGFβRII, and IL-7 in 7t15-TGFRswith ELISA.

FIG. 103 shows results of a 32Dβ cell proliferation assay with7t15-TGFRs or recombinant IL-15.

FIG. 104 is a line graph showing the chromatographic profile of7t15-TGFRs protein containing cell culture supernatant following bindingand elution on anti-TF antibody affinity column.

FIG. 105 shows 7t15-TGFRs before and after deglycosylation as analyzedusing reduced SDS-PAGE.

FIG. 106 shows ELISA detection of IL-7, IL-15 and TGFβRII in the7t15-TGFRs protein.

FIGS. 107A and 107B show spleen weight and the percentages of immunecell types in 7t15-TGFRs-treated and control-treated mice. FIG. 107Ashows spleen weight in mice treated with 7t15-TGFRs at various dosages,as compared to PBS control. FIG. 107B shows the percentage of CD4⁺ Tcells, CD8⁺ T cells, and NK cells in mice treated with 7t15-TGFRs atvarious dosages, as compared to PBS control.

FIGS. 108A and 108B show upregulation of CD44 expression of CD4⁺ andCD8⁺ T cells by 7t15-TGFRs in C57BL/6 mice.

FIGS. 109A and 109B show upregulation of Ki67 expression and Granzyme Bexpression of CD8⁺ T cells and NK Cells by 7t15-TGFRs in C57BL/6 mice.

FIG. 110 shows enhancement of cytotoxicity of splenocytes by 7t15-TGFRsin C57BL/6 mice.

FIG. 111 shows a schematic of the TGFRt15-21s137L construct.

FIG. 112 shows an additional schematic of the TGFRt15-21s137L construct.

FIG. 113 is a line graph showing the chromatographic profile ofTGFRt15-21s137L protein containing cell culture supernatant followingbinding and elution on anti-TF antibody affinity column.

FIG. 114 shows a schematic of the TGFRt15-TGFRs21 construct.

FIG. 115 shows an additional schematic of the TGFRt15-TGFRs21 construct.

FIG. 116 is a line graph showing the chromatographic profile ofTGFRt15-TGFRs21 protein containing cell culture supernatant followingbinding and elution on anti-TF antibody affinity column.

FIG. 117 shows TGFRt15-TGFRs21 before and after deglycosylation asanalyzed by reduced SDS-PAGE.

FIGS. 118A and 118B show detection of components of TGFRt15-TGFRs21using ELISA.

FIGS. 119A and 119B show the percentages and proliferation of CD4⁺ Tcells, CD8⁺ T cells, and natural killer (NK) cells present in the spleenof control-treated and TGFRt15-TGFRs21-treated mice.

FIG. 120 shows upregulation of Granzyme B expression of splenocytes inmice treated with TGFRt15-TGFRs21.

FIG. 121 shows enhancement of cytotoxicity of splenocytes byTGFRt15-TGFRs21 in C57BL/6 Mice.

FIG. 122 shows a schematic of the TGFRt15-TGFRs16 construct.

FIG. 123 shows an additional schematic of the TGFRt15-TGFRs16 construct.

FIG. 124 shows a schematic of the TGFRt15-TGFRs137L construct.

FIG. 125 shows an additional schematic of the TGFRt15-TGFRs137Lconstruct.

FIG. 126 shows changes in the surface phenotype of lymphocytepopulations after stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s.

FIG. 127 shows an increase in phospho-STAT4 and phospho-STAT5 levels inNK cells after stimulation with 18t15-12s.

FIGS. 128A-128C show in vivo stimulation of Tregs, NK cells, and CD8⁺ Tcells in ApoE^(−/−) mice fed with a Western diet and treated withTGFRt15-TGFRs.

FIGS. 129A-129C show immunostimulation in C57BL/6 mice followingtreatment with TGFRt15-TGFRs.

FIGS. 130A and 130B show in vivo induction of proliferation of NK cellsand CD8⁺ T cells in ApoE^(−/−) mice fed with a Western diet and treatedwith TGFRt15-TGFRs.

FIGS. 131A and 131B show enhancement of cytotoxicity of NK cellsfollowing treatment of NK cells with TGFRt15-TGFRs.

FIGS. 132A and 132B show enhancement of ADCC activity of NK cellsfollowing treatment of NK cells with TGFRt15-TGFRs.

FIGS. 133A-133H show antitumor activity of TGFRt15-TGFRs plus anti-TRP1antibody (TA99) in combination with chemotherapy in a melanoma mousemodel.

FIGS. 134A-134C show amelioration of the Western diet-inducedhyperglycemia in ApoE^(−/−) mice by TGFRt15-TGFRs.

FIG. 135 shows cell surface staining summarizing the differentiation ofNK cells into cytokine-induced memory like NK Cells (CIML-NK Cells)after stimulation with 18t15-12s and cultured in rhIL15.

FIG. 136 shows upregulation shows upregulation of CD44hi memory T cellsupon treatment with TGFRt15-TGFRs.

FIG. 137 shows a graph of Factor X (FX) activation following treatmentwith single-chain or multi-chain chimeric polypeptides.

FIG. 138 shows clotting time for a buffer with varying concentrations ofInnovin in a prothrombin time (PT) test.

FIG. 139 shows clotting time for multi-chain chimeric polypeptides in aPT Assay.

FIG. 140 shows clotting time of the multi-chain chimeric polypeptides ina PT assay when mixed with 32DB cells.

FIG. 141 shows clotting time of multi-chain chimeric polypeptides in aPT assay when mixed with human PBMC.

FIG. 142 shows binding of 7t15-21s137L (long version) and 7t15-21s137L(short version) to CD137 (4.1BB).

FIG. 143A-143D show detection of IL7, IL21, IL15, and 4.1BBL in7t15-21s137L (long version) by the respective antibodies using ELISA.

FIG. 144 shows IL-15 activity of 7t15-21s137L (long version) and7t15-21s137L (short version) as evaluated by a IL2Rαβγ-containing CTLL2cell proliferation assay.

DETAILED DESCRIPTION

Provided herein are multi-chain chimeric polypeptides that include: (a)a first chimeric polypeptide including: (i) a first target-bindingdomain; (ii) a soluble tissue factor domain; and (iii) a first domain ofa pair of affinity domains; and (b) a second chimeric polypeptideincluding: (i) a second domain of a pair of affinity domains; and (ii) asecond target-binding domain, where the first chimeric polypeptide andthe second chimeric polypeptide associate through the binding of thefirst domain and the second domain of the pair of affinity domains. Alsoprovided herein are compositions that include any of the multi-chainchimeric polypeptides described herein, nucleic acids that encode any ofthe multi-chain chimeric polypeptides described herein, and cells thatinclude any of the nucleic acids that encode any of the multi-chainchimeric polypeptides described herein. Also provided herein are methodsof stimulating an immune cell and methods of treating a subject in needthereof that include the use of any of the multi-chain chimericpolypeptides described herein. Also provided herein are methods ofproducing any of the multi-chain chimeric polypeptides described herein.

In some examples of any of the multi-chain chimeric polypeptidesdescribed herein the total length of first chimeric polypeptide and/orthe second chimeric polypeptide can each independently be about 50 aminoacids to about 3000 amino acids, about 50 amino acids to about 2500amino acids, about 50 amino acids to about 2000 amino acids, about 50amino acids to about 1500 amino acids, about 50 amino acids to about1000 amino acids, about 50 amino acids to about 950 amino acids, about50 amino acids to about 900 amino acids, about 50 amino acids to about850 amino acids, about 50 amino acids to about 800 amino acids, about 50amino acids to about 750 amino acids, about 50 amino acids to about 700amino acids, about 50 amino acids to about 650 amino acids, about 50amino acids to about 600 amino acids, about 50 amino acids to about 550amino acids, about 50 amino acids to about 500 amino acids, about 50amino acids to about 480 amino acids, about 50 amino acids to about 460amino acids, about 50 amino acids to about 440 amino acids, about 50amino acids to about 420 amino acids, about 50 amino acids to about 400amino acids, about 50 amino acids to about 380 amino acids, about 50amino acids to about 360 amino acids, about 50 amino acids to about 340amino acids, about 50 amino acids to about 320 amino acids, about 50amino acids to about 300 amino acids, about 50 amino acids to about 280amino acids, about 50 amino acids to about 260 amino acids, about 50amino acids to about 240 amino acids, about 50 amino acids to about 220amino acids, about 50 amino acids to about 200 amino acids, about 50amino acids to about 150 amino acids, about 50 amino acids to about 100amino acids, about 100 amino acids to about 3000 amino acids, about 100amino acids to about 2500 amino acids, about 100 amino acids to about2000 amino acids, about 100 amino acids to about 1500 amino acids, about100 amino acids to about 1000 amino acids, about 100 amino acids toabout 950 amino acids, about 100 amino acids to about 900 amino acids,about 100 amino acids to about 850 amino acids, about 100 amino acids toabout 800 amino acids, about 100 amino acids to about 750 amino acids,about 100 amino acids to about 700 amino acids, about 100 amino acids toabout 650 amino acids, about 100 amino acids to about 600 amino acids,about 100 amino acids to about 550 amino acids, about 100 amino acids toabout 500 amino acids, about 100 amino acids to about 480 amino acids,about 100 amino acids to about 460 amino acids, about 100 amino acids toabout 440 amino acids, about 100 amino acids to about 420 amino acids,about 100 amino acids to about 400 amino acids, about 100 amino acids toabout 380 amino acids, about 100 amino acids to about 360 amino acids,about 100 amino acids to about 340 amino acids, about 100 amino acids toabout 320 amino acids, about 100 amino acids to about 300 amino acids,about 100 amino acids to about 280 amino acids, about 100 amino acids toabout 260 amino acids, about 100 amino acids to about 240 amino acids,about 100 amino acids to about 220 amino acids, about 100 amino acids toabout 200 amino acids, about 100 amino acids to about 150 amino acids,about 150 amino acids to about 3000 amino acids, about 150 amino acidsto about 2500 amino acids, about 150 amino acids to about 2000 aminoacids, about 150 amino acids to about 1500 amino acids, about 150 aminoacids to about 1000 amino acids, about 150 amino acids to about 950amino acids, about 150 amino acids to about 900 amino acids, about 150amino acids to about 850 amino acids, about 150 amino acids to about 800amino acids, about 150 amino acids to about 750 amino acids, about 150amino acids to about 700 amino acids, about 150 amino acids to about 650amino acids, about 150 amino acids to about 600 amino acids, about 150amino acids to about 550 amino acids, about 150 amino acids to about 500amino acids, about 150 amino acids to about 480 amino acids, about 150amino acids to about 460 amino acids, about 150 amino acids to about 440amino acids, about 150 amino acids to about 420 amino acids, about 150amino acids to about 400 amino acids, about 150 amino acids to about 380amino acids, about 150 amino acids to about 360 amino acids, about 150amino acids to about 340 amino acids, about 150 amino acids to about 320amino acids, about 150 amino acids to about 300 amino acids, about 150amino acids to about 280 amino acids, about 150 amino acids to about 260amino acids, about 150 amino acids to about 240 amino acids, about 150amino acids to about 220 amino acids, about 150 amino acids to about 200amino acids, about 200 amino acids to about 3000 amino acids, about 200amino acids to about 2500 amino acids, about 200 amino acids to about2000 amino acids, about 200 amino acids to about 1500 amino acids, about200 amino acids to about 1000 amino acids, about 200 amino acids toabout 950 amino acids, about 200 amino acids to about 900 amino acids,about 200 amino acids to about 850 amino acids, about 200 amino acids toabout 800 amino acids, about 200 amino acids to about 750 amino acids,about 200 amino acids to about 700 amino acids, about 200 amino acids toabout 650 amino acids, about 200 amino acids to about 600 amino acids,about 200 amino acids to about 550 amino acids, about 200 amino acids toabout 500 amino acids, about 200 amino acids to about 480 amino acids,about 200 amino acids to about 460 amino acids, about 200 amino acids toabout 440 amino acids, about 200 amino acids to about 420 amino acids,about 200 amino acids to about 400 amino acids, about 200 amino acids toabout 380 amino acids, about 200 amino acids to about 360 amino acids,about 200 amino acids to about 340 amino acids, about 200 amino acids toabout 320 amino acids, about 200 amino acids to about 300 amino acids,about 200 amino acids to about 280 amino acids, about 200 amino acids toabout 260 amino acids, about 200 amino acids to about 240 amino acids,about 200 amino acids to about 220 amino acids, about 220 amino acids toabout 3000 amino acids, about 220 amino acids to about 2500 amino acids,about 220 amino acids to about 2000 amino acids, about 220 amino acidsto about 1500 amino acids, about 220 amino acids to about 1000 aminoacids, about 220 amino acids to about 950 amino acids, about 220 aminoacids to about 900 amino acids, about 220 amino acids to about 850 aminoacids, about 220 amino acids to about 800 amino acids, about 220 aminoacids to about 750 amino acids, about 220 amino acids to about 700 aminoacids, about 220 amino acids to about 650 amino acids, about 220 aminoacids to about 600 amino acids, about 220 amino acids to about 550 aminoacids, about 220 amino acids to about 500 amino acids, about 220 aminoacids to about 480 amino acids, about 220 amino acids to about 460 aminoacids, about 220 amino acids to about 440 amino acids, about 220 aminoacids to about 420 amino acids, about 220 amino acids to about 400 aminoacids, about 220 amino acids to about 380 amino acids, about 220 aminoacids to about 360 amino acids, about 220 amino acids to about 340 aminoacids, about 220 amino acids to about 320 amino acids, about 220 aminoacids to about 300 amino acids, about 220 amino acids to about 280 aminoacids, about 220 amino acids to about 260 amino acids, about 220 aminoacids to about 240 amino acids, about 240 amino acids to about 3000amino acids, about 240 amino acids to about 2500 amino acids, about 240amino acids to about 2000 amino acids, about 240 amino acids to about1500 amino acids, about 240 amino acids to about 1000 amino acids, about240 amino acids to about 950 amino acids, about 240 amino acids to about900 amino acids, about 240 amino acids to about 850 amino acids, about240 amino acids to about 800 amino acids, about 240 amino acids to about750 amino acids, about 240 amino acids to about 700 amino acids, about240 amino acids to about 650 amino acids, about 240 amino acids to about600 amino acids, about 240 amino acids to about 550 amino acids, about240 amino acids to about 500 amino acids, about 240 amino acids to about480 amino acids, about 240 amino acids to about 460 amino acids, about240 amino acids to about 440 amino acids, about 240 amino acids to about420 amino acids, about 240 amino acids to about 400 amino acids, about240 amino acids to about 380 amino acids, about 240 amino acids to about360 amino acids, about 240 amino acids to about 340 amino acids, about240 amino acids to about 320 amino acids, about 240 amino acids to about300 amino acids, about 240 amino acids to about 280 amino acids, about240 amino acids to about 260 amino acids, about 260 amino acids to about3000 amino acids, about 260 amino acids to about 2500 amino acids, about260 amino acids to about 2000 amino acids, about 260 amino acids toabout 1500 amino acids, about 260 amino acids to about 1000 amino acids,about 260 amino acids to about 950 amino acids, about 260 amino acids toabout 900 amino acids, about 260 amino acids to about 850 amino acids,about 260 amino acids to about 800 amino acids, about 260 amino acids toabout 750 amino acids, about 260 amino acids to about 700 amino acids,about 260 amino acids to about 650 amino acids, about 260 amino acids toabout 600 amino acids, about 260 amino acids to about 550 amino acids,about 260 amino acids to about 500 amino acids, about 260 amino acids toabout 480 amino acids, about 260 amino acids to about 460 amino acids,about 260 amino acids to about 440 amino acids, about 260 amino acids toabout 420 amino acids, about 260 amino acids to about 400 amino acids,about 260 amino acids to about 380 amino acids, about 260 amino acids toabout 360 amino acids, about 260 amino acids to about 340 amino acids,about 260 amino acids to about 320 amino acids, about 260 amino acids toabout 300 amino acids, about 260 amino acids to about 280 amino acids,about 280 amino acids to about 3000 amino acids, about 280 amino acidsto about 2500 amino acids, about 280 amino acids to about 2000 aminoacids, about 280 amino acids to about 1500 amino acids, about 280 aminoacids to about 1000 amino acids, about 280 amino acids to about 950amino acids, about 280 amino acids to about 900 amino acids, about 280amino acids to about 850 amino acids, about 280 amino acids to about 800amino acids, about 280 amino acids to about 750 amino acids, about 280amino acids to about 700 amino acids, about 280 amino acids to about 650amino acids, about 280 amino acids to about 600 amino acids, about 280amino acids to about 550 amino acids, about 280 amino acids to about 500amino acids, about 280 amino acids to about 480 amino acids, about 280amino acids to about 460 amino acids, about 280 amino acids to about 440amino acids, about 280 amino acids to about 420 amino acids, about 280amino acids to about 400 amino acids, about 280 amino acids to about 380amino acids, about 280 amino acids to about 360 amino acids, about 280amino acids to about 340 amino acids, about 280 amino acids to about 320amino acids, about 280 amino acids to about 300 amino acids, about 300amino acids to about 3000 amino acids, about 300 amino acids to about2500 amino acids, about 300 amino acids to about 2000 amino acids, about300 amino acids to about 1500 amino acids, about 300 amino acids toabout 1000 amino acids, about 300 amino acids to about 950 amino acids,about 300 amino acids to about 900 amino acids, about 300 amino acids toabout 850 amino acids, about 300 amino acids to about 800 amino acids,about 300 amino acids to about 750 amino acids, about 300 amino acids toabout 700 amino acids, about 300 amino acids to about 650 amino acids,about 300 amino acids to about 600 amino acids, about 300 amino acids toabout 550 amino acids, about 300 amino acids to about 500 amino acids,about 300 amino acids to about 480 amino acids, about 300 amino acids toabout 460 amino acids, about 300 amino acids to about 440 amino acids,about 300 amino acids to about 420 amino acids, about 300 amino acids toabout 400 amino acids, about 300 amino acids to about 380 amino acids,about 300 amino acids to about 360 amino acids, about 300 amino acids toabout 340 amino acids, about 300 amino acids to about 320 amino acids,about 320 amino acids to about 3000 amino acids, about 320 amino acidsto about 2500 amino acids, about 320 amino acids to about 2000 aminoacids, about 320 amino acids to about 1500 amino acids, about 320 aminoacids to about 1000 amino acids, about 320 amino acids to about 950amino acids, about 320 amino acids to about 900 amino acids, about 320amino acids to about 850 amino acids, about 320 amino acids to about 800amino acids, about 320 amino acids to about 750 amino acids, about 320amino acids to about 700 amino acids, about 320 amino acids to about 650amino acids, about 320 amino acids to about 600 amino acids, about 320amino acids to about 550 amino acids, about 320 amino acids to about 500amino acids, about 320 amino acids to about 480 amino acids, about 320amino acids to about 460 amino acids, about 320 amino acids to about 440amino acids, about 320 amino acids to about 420 amino acids, about 320amino acids to about 400 amino acids, about 320 amino acids to about 380amino acids, about 320 amino acids to about 360 amino acids, about 320amino acids to about 340 amino acids, about 340 amino acids to about3000 amino acids, about 340 amino acids to about 2500 amino acids, about340 amino acids to about 2000 amino acids, about 340 amino acids toabout 1500 amino acids, about 340 amino acids to about 1000 amino acids,about 340 amino acids to about 950 amino acids, about 340 amino acids toabout 900 amino acids, about 340 amino acids to about 850 amino acids,about 340 amino acids to about 800 amino acids, about 340 amino acids toabout 750 amino acids, about 340 amino acids to about 700 amino acids,about 340 amino acids to about 650 amino acids, about 340 amino acids toabout 600 amino acids, about 340 amino acids to about 550 amino acids,about 340 amino acids to about 500 amino acids, about 340 amino acids toabout 480 amino acids, about 340 amino acids to about 460 amino acids,about 340 amino acids to about 440 amino acids, about 340 amino acids toabout 420 amino acids, about 340 amino acids to about 400 amino acids,about 340 amino acids to about 380 amino acids, about 340 amino acids toabout 360 amino acids, about 360 amino acids to about 3000 amino acids,about 360 amino acids to about 2500 amino acids, about 360 amino acidsto about 2000 amino acids, about 360 amino acids to about 1500 aminoacids, about 360 amino acids to about 1000 amino acids, about 360 aminoacids to about 950 amino acids, about 360 amino acids to about 900 aminoacids, about 360 amino acids to about 850 amino acids, about 360 aminoacids to about 800 amino acids, about 360 amino acids to about 750 aminoacids, about 360 amino acids to about 700 amino acids, about 360 aminoacids to about 650 amino acids, about 360 amino acids to about 600 aminoacids, about 360 amino acids to about 550 amino acids, about 360 aminoacids to about 500 amino acids, about 360 amino acids to about 480 aminoacids, about 360 amino acids to about 460 amino acids, about 360 aminoacids to about 440 amino acids, about 360 amino acids to about 420 aminoacids, about 360 amino acids to about 400 amino acids, about 360 aminoacids to about 380 amino acids, about 380 amino acids to about 3000amino acids, about 380 amino acids to about 2500 amino acids, about 380amino acids to about 2000 amino acids, about 380 amino acids to about1500 amino acids, about 380 amino acids to about 1000 amino acids, about380 amino acids to about 950 amino acids, about 380 amino acids to about900 amino acids, about 380 amino acids to about 850 amino acids, about380 amino acids to about 800 amino acids, about 380 amino acids to about750 amino acids, about 380 amino acids to about 700 amino acids, about380 amino acids to about 650 amino acids, about 380 amino acids to about600 amino acids, about 380 amino acids to about 550 amino acids, about380 amino acids to about 500 amino acids, about 380 amino acids to about480 amino acids, about 380 amino acids to about 460 amino acids, about380 amino acids to about 440 amino acids, about 380 amino acids to about420 amino acids, about 380 amino acids to about 400 amino acids, about400 amino acids to about 3000 amino acids, about 400 amino acids toabout 2500 amino acids, about 400 amino acids to about 2000 amino acids,about 400 amino acids to about 1500 amino acids, about 400 amino acidsto about 1000 amino acids, about 400 amino acids to about 950 aminoacids, about 400 amino acids to about 900 amino acids, about 400 aminoacids to about 850 amino acids, about 400 amino acids to about 800 aminoacids, about 400 amino acids to about 750 amino acids, about 400 aminoacids to about 700 amino acids, about 400 amino acids to about 650 aminoacids, about 400 amino acids to about 600 amino acids, about 400 aminoacids to about 550 amino acids, about 400 amino acids to about 500 aminoacids, about 400 amino acids to about 480 amino acids, about 400 aminoacids to about 460 amino acids, about 400 amino acids to about 440 aminoacids, about 400 amino acids to about 420 amino acids, about 420 aminoacids to about 3000 amino acids, about 420 amino acids to about 2500amino acids, about 420 amino acids to about 2000 amino acids, about 420amino acids to about 1500 amino acids, about 420 amino acids to about1000 amino acids, about 420 amino acids to about 950 amino acids, about420 amino acids to about 900 amino acids, about 420 amino acids to about850 amino acids, about 420 amino acids to about 800 amino acids, about420 amino acids to about 750 amino acids, about 420 amino acids to about700 amino acids, about 420 amino acids to about 650 amino acids, about420 amino acids to about 600 amino acids, about 420 amino acids to about550 amino acids, about 420 amino acids to about 500 amino acids, about420 amino acids to about 480 amino acids, about 420 amino acids to about460 amino acids, about 420 amino acids to about 440 amino acids, about440 amino acids to about 3000 amino acids, about 440 amino acids toabout 2500 amino acids, about 440 amino acids to about 2000 amino acids,about 440 amino acids to about 1500 amino acids, about 440 amino acidsto about 1000 amino acids, about 440 amino acids to about 950 aminoacids, about 440 amino acids to about 900 amino acids, about 440 aminoacids to about 850 amino acids, about 440 amino acids to about 800 aminoacids, about 440 amino acids to about 750 amino acids, about 440 aminoacids to about 700 amino acids, about 440 amino acids to about 650 aminoacids, about 440 amino acids to about 600 amino acids, about 440 aminoacids to about 550 amino acids, about 440 amino acids to about 500 aminoacids, about 440 amino acids to about 480 amino acids, about 440 aminoacids to about 460 amino acids, about 460 amino acids to about 3000amino acids, about 460 amino acids to about 2500 amino acids, about 460amino acids to about 2000 amino acids, about 460 amino acids to about1500 amino acids, about 460 amino acids to about 1000 amino acids, about460 amino acids to about 950 amino acids, about 460 amino acids to about900 amino acids, about 460 amino acids to about 850 amino acids, about460 amino acids to about 800 amino acids, about 460 amino acids to about750 amino acids, about 460 amino acids to about 700 amino acids, about460 amino acids to about 650 amino acids, about 460 amino acids to about600 amino acids, about 460 amino acids to about 550 amino acids, about460 amino acids to about 500 amino acids, about 460 amino acids to about480 amino acids, about 480 amino acids to about 3000 amino acids, about480 amino acids to about 2500 amino acids, about 480 amino acids toabout 2000 amino acids, about 480 amino acids to about 1500 amino acids,about 480 amino acids to about 1000 amino acids, about 480 amino acidsto about 950 amino acids, about 480 amino acids to about 900 aminoacids, about 480 amino acids to about 850 amino acids, about 480 aminoacids to about 800 amino acids, about 480 amino acids to about 750 aminoacids, about 480 amino acids to about 700 amino acids, about 480 aminoacids to about 650 amino acids, about 480 amino acids to about 600 aminoacids, about 480 amino acids to about 550 amino acids, about 480 aminoacids to about 500 amino acids, about 500 amino acids to about 3000amino acids, about 500 amino acids to about 2500 amino acids, about 500amino acids to about 2000 amino acids, about 500 amino acids to about1500 amino acids, about 500 amino acids to about 1000 amino acids, about500 amino acids to about 950 amino acids, about 500 amino acids to about900 amino acids, about 500 amino acids to about 850 amino acids, about500 amino acids to about 800 amino acids, about 500 amino acids to about750 amino acids, about 500 amino acids to about 700 amino acids, about500 amino acids to about 650 amino acids, about 500 amino acids to about600 amino acids, about 500 amino acids to about 550 amino acids, about550 amino acids to about 3000 amino acids, about 550 amino acids toabout 2500 amino acids, about 550 amino acids to about 2000 amino acids,about 550 amino acids to about 1500 amino acids, about 550 amino acidsto about 1000 amino acids, about 550 amino acids to about 950 aminoacids, about 550 amino acids to about 900 amino acids, about 550 aminoacids to about 850 amino acids, about 550 amino acids to about 800 aminoacids, about 550 amino acids to about 750 amino acids, about 550 aminoacids to about 700 amino acids, about 550 amino acids to about 650 aminoacids, about 550 amino acids to about 600 amino acids, about 600 aminoacids to about 3000 amino acids, about 600 amino acids to about 2500amino acids, about 600 amino acids to about 2000 amino acids, about 600amino acids to about 1500 amino acids, about 600 amino acids to about1000 amino acids, about 600 amino acids to about 950 amino acids, about600 amino acids to about 900 amino acids, about 600 amino acids to about850 amino acids, about 600 amino acids to about 800 amino acids, about600 amino acids to about 750 amino acids, about 600 amino acids to about700 amino acids, about 600 amino acids to about 650 amino acids, about650 amino acids to about 3000 amino acids, about 650 amino acids toabout 2500 amino acids, about 650 amino acids to about 2000 amino acids,about 650 amino acids to about 1500 amino acids, about 650 amino acidsto about 1000 amino acids, about 650 amino acids to about 950 aminoacids, about 650 amino acids to about 900 amino acids, about 650 aminoacids to about 850 amino acids, about 650 amino acids to about 800 aminoacids, about 650 amino acids to about 750 amino acids, about 650 aminoacids to about 700 amino acids, about 700 amino acids to about 3000amino acids, about 700 amino acids to about 2500 amino acids, about 700amino acids to about 2000 amino acids, about 700 amino acids to about1500 amino acids, about 700 amino acids to about 1000 amino acids, about700 amino acids to about 950 amino acids, about 700 amino acids to about900 amino acids, about 700 amino acids to about 850 amino acids, about700 amino acids to about 800 amino acids, about 700 amino acids to about750 amino acids, about 750 amino acids to about 3000 amino acids, about750 amino acids to about 2500 amino acids, about 750 amino acids toabout 2000 amino acids, about 750 amino acids to about 1500 amino acids,about 750 amino acids to about 1000 amino acids, about 750 amino acidsto about 950 amino acids, about 750 amino acids to about 900 aminoacids, about 750 amino acids to about 850 amino acids, about 750 aminoacids to about 800 amino acids, about 800 amino acids to about 3000amino acids, about 800 amino acids to about 2500 amino acids, about 800amino acids to about 2000 amino acids, about 800 amino acids to about1500 amino acids, about 800 amino acids to about 1000 amino acids, about800 amino acids to about 950 amino acids, about 800 amino acids to about900 amino acids, about 800 amino acids to about 850 amino acids, about850 amino acids to about 3000 amino acids, about 850 amino acids toabout 2500 amino acids, about 850 amino acids to about 2000 amino acids,about 850 amino acids to about 1500 amino acids, about 850 amino acidsto about 1000 amino acids, about 850 amino acids to about 950 aminoacids, about 850 amino acids to about 900 amino acids, about 900 aminoacids to about 3000 amino acids, about 900 amino acids to about 2500amino acids, about 900 amino acids to about 2000 amino acids, about 900amino acids to about 1500 amino acids, about 900 amino acids to about1000 amino acids, about 900 amino acids to about 950 amino acids, about950 amino acids to about 3000 amino acids, about 950 amino acids toabout 2500 amino acids, about 950 amino acids to about 2000 amino acids,about 950 amino acids to about 1500 amino acids, about 950 amino acidsto about 1000 amino acids, about 1000 amino acids to about 3000 aminoacids, about 1000 amino acids to about 2500 amino acids, about 1000amino acids to about 2000 amino acids, about 1000 amino acids to about1500 amino acids, about 1500 amino acids to about 3000 amino acids,about 1500 amino acids to about 2500 amino acids, about 1500 amino acidsto about 2000 amino acids, about 2000 amino acids to about 3000 aminoacids, about 2000 amino acids to about 2500 amino acids, or about 2500amino acids to about 3000 amino acids. Diagrams of exemplary multi-chainchimeric polypeptides provided herein are depicted in FIGS. 1 and 2 .

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain (e.g., any of thefirst target-binding domains described herein) and the soluble tissuefactor domain (e.g., any of the exemplary soluble tissue factor domainsdescribed herein) directly abut each other in the first chimericpolypeptide. In some embodiments of any of the multi-chain chimericpolypeptides described herein, the first chimeric polypeptide furthercomprises a linker sequence (e.g., any of the exemplary linker sequencesdescribed herein or known in the art) between the first target-bindingdomain (e.g., any of the exemplary first target-binding domainsdescribed herein) and the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains described herein) in the firstchimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains described herein) and the firstdomain of the pair of affinity domains (e.g., any of the exemplary firstdomains of any of the exemplary pairs of affinity domains describedherein) directly abut each other in the first chimeric polypeptide. Insome embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first chimeric polypeptide further comprises alinker sequence (e.g., any of the exemplary linker sequences describedherein or known in the art) between the soluble tissue factor domain(e.g., any of the exemplary soluble tissue factor domains describedherein) and the first domain of the pair of affinity domains (e.g., anyof the exemplary first domains of any of the exemplary pairs of affinitydomains described herein) in the first chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the second domain of the pair of affinity domains(e.g., any of the exemplary second domains of any of the exemplary pairsof affinity domains described herein) and the second target-bindingdomain (e.g., any of the exemplary second target-binding domainsdescribed herein) directly abut each other in the second chimericpolypeptide. In some embodiments of any of the multi-chain chimericpolypeptides described herein, the second chimeric polypeptide furthercomprises a linker sequence (e.g., any of the exemplary linker sequencesdescribed herein or known in the art) between the second domain of thepair of affinity domains (e.g., any of the exemplary second domains ofany of the exemplary pairs of affinity domains described herein) and thesecond target-binding domain (e.g., any of the exemplary secondtarget-binding domains described herein) in the second chimericpolypeptide.

Non-limiting aspects of these chimeric polypeptides, nucleic acids,vectors, cells, and methods are described below, and can be used in anycombination without limitation. Additional aspects of these chimericpolypeptides, nucleic acids, vectors, cells, and methods are known inthe art.

Tissue Factor

Human tissue factor is a 263 amino-acid transmembrane protein containingthree domains: (1) a 219-amino acid N-terminal extracellular domain(residues 1-219); (2) a 22-amino acid transmembrane domain (residues220-242); and (3) a 21-amino acid cytoplasmic C-terminal tail (residues242-263) ((UniProtKB Identifier Number: P13726). The cytoplasmic tailcontains two phosphorylation sites at Ser253 and Ser258, and oneS-palmitoylation site at Cys245. Deletion or mutation of the cytoplasmicdomain was not found to affect tissue factor coagulation activity.Tissue factor has one S-palmitoylation site in the intracellular domainof the protein at Cys245. The Cys245 is located at the amino acidterminus of the intracellular domain and close to the membrane surface.The tissue factor transmembrane domain is composed of a single-spanningα-helix.

The extracellular domain of tissue factor, composed of two fibronectintype III domains, is connected to the transmembrane domain through asix-amino acid linker. This linker provides conformational flexibilityto decouple the tissue factor extracellular domain from itstransmembrane and cytoplasmic domains. Each tissue factor fibronectintype III module is composed of two overlapping (3 sheets with the topsheet domain containing three antiparallel β-strands and the bottomsheet containing four β-strands. The β-strands are connected by β-loopsbetween strand βA and βB, βC and βD, and βE and βF, all of which areconserved in conformation in the two modules. There are three shortα-helix segments connecting the β-strands. A unique feature of tissuefactor is a 17-amino acid β-hairpin between strand β10 and strand β11,which is not a common element of the fibronectin superfamily. TheN-terminal domain also contains a 12 amino acid loop between β6F and β7Gthat is not present in the C-terminal domain and is unique to tissuefactor. Such a fibronectin type III domain structure is a feature of theimmunoglobulin-like family of protein folds and is conserved among awide variety of extracellular proteins.

The zymogen FVII is rapidly converted to FVIIa by limited proteolysisonce it binds to tissue to form the active tissue factor-FVIIa complex.The FVIIa, which circulates as an enzyme at a concentration ofapproximately 0.1 nM (1% of plasma FVII), can also bind directly totissue factor. The allosteric interaction between tissue factor andFVIIa on the tissue factor-FVIIa complex greatly increases the enzymaticactivity of FVIIa: an approximate 20- to 100-fold increase in the rateof hydrolysis of small, chromogenic peptidyl substrates, and nearly amillion-fold increase in the rate of activation of the naturalmacromolecular substrates FIX and FX. In concert with allostericactivation of the active site of FVIIa upon binding to tissue factor,the formation of tissue factor-FVIIa complex on phospholipid bilayer(i.e., upon exposure of phosphatidyl-L-serine on membrane surfaces)increases the rate of FIX or FX activation, in a Ca²⁺-dependent manner,an additional 1,000-fold. The roughly million-fold overall increase inFX activation by tissue factor-FVIIa-phospholipid complex relative tofree FVIIa is a critical regulatory point for the coagulation cascade.

FVII is a ˜50 kDa, single-chain polypeptide consisting of 406 amino acidresidues, with an N-terminal γ-carboxyglutamate-rich (GLA) domain, twoepidermal growth factor-like domains (EGF1 and EFG2), and a C-terminalserine protease domain. FVII is activated to FVIIa by a specificproteolytic cleavage of the Ile-¹⁵⁴-Arg¹⁵² bond in the short linkerregion between the EGF2 and the protease domain. This cleavage resultsin the light and heavy chains being held together by a single disulfidebond of Cys¹³⁵ and Cys²⁶². FVIIa binds phospholipid membrane in aCa²⁺-dependent manner through its N-terminal GLA-domain. ImmediatelyC-terminal to the GLA domain is an aromatic stack and two EGF domains.The aromatic stack connects the GLA to EGF1 domain which binds a singleCa²⁺ ion. Occupancy of this Ca²⁺-binding site increases FVIIa amidolyticactivity and tissue factor association. The catalytic triad consist ofHis¹⁹⁸, Asp²⁴², and Ser³⁴⁴, and binding of a single Ca²⁺ ion within theFVIIa protease domain is critical for its catalytic activity.Proteolytic activation of FVII to FVIIa frees the newly formed aminoterminus at Ile¹⁵³ to fold back and be inserted into the activationpocket forming a salt bridge with the carboxylate of Asp³⁴³ to generatethe oxyanion hole. Formation of this salt bridge is critical for FVIIaactivity. However, oxyanion hole formation does not occur in free FVIIaupon proteolytic activation. As a result, FVIIa circulates in azymogen-like state that is poorly recognized by plasma proteaseinhibitors, allowing it to circulate with a half-life of approximately90 minutes.

Tissue factor-mediated positioning of the FVIIa active site above themembrane surface is important for FVIIa towards cognate substrates. FreeFVIIa adopts a stable, extended structure when bound to the membranewith its active site positioned ˜80 Å above the membrane surface. UponFVIIa binding to tissue factor, the FVa active site is repositioned ˜6 Åcloser to the membrane. This modulation may aid in a proper alignment ofthe FVIIa catalytic triad with the target substrate cleavage site. UsingGLA-domainless FVIIa, it has been shown that the active site was stillpositioned a similar distance above the membrane, demonstrating thattissue factor is able to fully support FVIIa active site positioningeven in the absence of FVIIa-membrane interaction. Additional datashowed that tissue factor supported full FVIIa proteolytic activity aslong as the tissue factor extracellular domain was tethered in some wayto the membrane surface. However, raising the active site of FVIIagreater than 80A above the membrane surface greatly reduced the abilityof the tissue factor-FVIIa complex to activate FX but did not diminishtissue factor-FVIIa amidolytic activity.

Alanine scanning mutagenesis has been used to assess the role ofspecific amino acid side chains in the tissue factor extracellulardomain for interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30): 19399-19403,1994). Alanine substitution identified a limited number of residuepositions at which alanine replacements cause 5- to 10-fold loweraffinity for FVIIa binding. Most of these residue side chains were foundto be well-exposed to solvent in the crystal structure, concordant withmacromolecular ligand interaction. The FVIIa ligand-binding site islocated over an extensive region at the boundary between the twomodules. In the C-module, residues Arg¹³⁵ and Phe¹⁴⁰ located on theprotruding B-C loop provide an independent contact with FVIIa. Leu¹³³ islocated at the base of the fingerlike structure and packed into thecleft between the two modules. This provides continuity to a majorcluster of important binding residues consisting of Lys²⁰, Thr⁶⁰ ,Asp⁵⁸, and Ile²². Thr⁶⁰ is only partially solvent-exposed and may play alocal structural role rather than making a significant contact withligand. The binding site extends onto the concave side of theintermodule angle involving Glu²⁴ and Gln¹¹⁰, and potentially the moredistant residue Val²⁰⁷. The binding region extends from Asp58 onto aconvex surface area formed by Lys⁴⁸, Lys⁴⁶, Gln³⁷, Asp⁴⁴, and Trp⁴⁵.Trp⁴⁵ and Asp⁴⁴ do not interact independently with FVIIa, indicatingthat the mutational effect at the Trp⁴⁵ position may reflect astructural importance of this side chain for the local packing of theadjacent Asp⁴⁴ and Gln³⁷ side chain. The interactive area furtherincludes two surface-exposed aromatic residues, Phe⁷⁶ and Tyr⁷⁸, whichform part of the hydrophobic cluster in the N-module.

The known physiologic substrates of tissue factor-FVIIa are FVII, FIX,and FX and certain proteinase-activated receptors. Mutational analysishas identified a number of residues that, when mutated, support fullFVIIa amidolytic activity towards small peptidyl substrates but aredeficient in their ability to support macromolecular substrate (i.e.,FVII, FIX, and FX) activation (Ruf et al., J Biol Chem 267(31):22206-22210, 1992; Ruf et al., J Biol Chem 267(9): 6375-6381, 1992;Huang et al., J Biol Chem 271(36): 21752-21757, 1996; Kirchhofer et al.,Biochemistry 39(25): 7380-7387, 2000). The tissue factor loop region atresidues 159-165, and residues in or adjacent to this flexible loop havebeen shown to be critical for the proteolytic activity of the tissuefactor-FVIIa complex. This defines the proposed substrate-bindingexosite region of tissue factor that is quite distant from the FVIIaactive site. A substitution of the glycine residue by a marginallybulkier residue alanine, significantly impairs tissue factor-FVIIaproteolytic activity. This suggests that the flexibility afforded byglycine is critical for the loop of residues 159-165 for tissue factormacromolecular substrate recognition.

The residues Lys¹⁶⁵ and Lys¹⁶⁶ have also been demonstrated to beimportant for substrate recognition and binding. Mutation of either ofthese residues to alanine results in a significant decrease in thetissue factor co-factor function. Lys¹⁶⁵ and Lys¹⁶⁶ face away from eachother, with Lys¹⁶⁵ pointing towards FVIIa in most tissue factor-FVIIastructures, and Lys¹⁶⁶ pointing into the substrate binding exositeregion in the crystal structure. Putative salt bridge formation betweenLys¹⁶⁵ of and Gla³⁵ of FVIIa would support the notion that tissue factorinteraction with the GLA domain of FVIIa modulates substraterecognition. These results suggest that the C-terminal portion of thetissue factor ectodomain directly interacts with the GLA-domain, thepossible adjacent EGF1 domains, of FIX and FX, and that the presence ofthe FVIIa GLA-domain may modulate these interactions either directly orindirectly.

Soluble Tissue Factor Domain

In some embodiments of any of the polypeptides, compositions, or methodsdescribed herein, the soluble tissue factor domain can be a wildtypetissue factor polypeptide lacking the signal sequence, the transmembranedomain, and the intracellular domain. In some examples, the solubletissue factor domain can be a tissue factor mutant, wherein a wildtypetissue factor polypeptide lacking the signal sequence, the transmembranedomain, and the intracellular domain, and has been further modified atselected amino acids. In some examples, the soluble tissue factor domaincan be a soluble human tissue factor domain. In some examples, thesoluble tissue factor domain can be a soluble mouse tissue factordomain. In some examples, the soluble tissue factor domain can be asoluble rat tissue factor domain. Non-limiting examples of soluble humantissue factor domains, a mouse soluble tissue factor domain, a ratsoluble tissue factor domain, and mutant soluble tissue factor domainsare shown below.

Exemplary Soluble Human Tissue Factor Domain (SEQ ID NO: 1)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFREExemplary Nucleic Acid Encoding Soluble Human Tissue Factor Domain (SEQID NO: 2) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG ExemplaryMutant Soluble Human Tissue Factor Domain (SEQ ID NO: 3)SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFREExemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO: 4)SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFREExemplary Soluble Mouse Tissue Factor Domain (SEQ ID NO: 5)agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnapkflpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge Exemplary Soluble Rat Tissue Factor Domain (SEQID NO: 6) agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtkigqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge

In some embodiments, a soluble tissue factor domain can include asequence that is at least 70% identical, at least 72% identical, atleast 74% identical, at least 76% identical, at least 78% identical, atleast 80% identical, at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical to SEQ IDNO: 1, 3, 4, 5, or 6. In some embodiments, a soluble tissue factordomain can include a sequence of SEQ ID NO: 1, 3, 4, 5, or 6, with oneto twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20) amino acids removed from its N-terminusand/or one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids removed from itsC-terminus.

As can be appreciated in the art, one skilled in the art wouldunderstand that mutation of amino acids that are conserved betweendifferent mammalian species is more likely to decrease the activityand/or structural stability of the protein, while mutation of aminoacids that are not conserved between different mammalian species is lesslikely to decrease the activity and/or structural stability of theprotein.

In some examples of any of the multi-chain chimeric polypeptidesdescribed herein, the soluble tissue factor domain is not capable ofbinding to Factor VIIa. In some examples of any of the multi-chainchimeric polypeptides described herein, the soluble tissue factor domaindoes not convert inactive Factor X into Factor Xa. In some embodimentsof any of the multi-chain chimeric polypeptides described herein, themulti-chain chimeric polypeptide does not stimulate blood coagulation ina mammal.

In some examples, the soluble tissue factor domain can be a solublehuman tissue factor domain. In some embodiments, the soluble tissuefactor domain can be a soluble mouse tissue factor domain. In someembodiments, the soluble tissue factor domain can be a soluble rattissue factor domain.

In some examples, the soluble tissue factor domain does not include oneor more (e.g., two, three, four, five, six, or seven) of: a lysine at anamino acid position that corresponds to amino acid position 20 of maturewildtype human tissue factor protein; an isoleucine at an amino acidposition that corresponds to amino acid position 22 of mature wildtypehuman tissue factor protein; a tryptophan at an amino acid position thatcorresponds to amino acid position 45 of mature wildtype human tissuefactor protein; an aspartic acid at an amino acid position thatcorresponds to amino acid position 58 of mature wildtype human tissuefactor protein; a tyrosine at an amino acid position that corresponds toamino acid position 94 of mature wildtype human tissue factor protein;an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and aphenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein. In someembodiments, the mutant soluble tissue factor possesses the amino acidsequence of SEQ ID NO: 3 or SEQ ID NO: 4.

In some examples, the soluble tissue factor domain can be encoded by anucleic acid including a sequence that is at least 70% identical, atleast 72% identical, at least 74% identical, at least 76% identical, atleast 78% identical, at least 80% identical, at least 82% identical, atleast 84% identical, at least 86% identical, at least 88% identical, atleast 90% identical, at least 92% identical, at least 94% identical, atleast 96% identical, at least 98% identical, at least 99% identical, or100% identical to SEQ ID NO: 2.

In some embodiments, the soluble tissue factor domain can have a totallength of about 20 amino acids to about 220 amino acids, about 20 aminoacids to about 215 amino acids, about 20 amino acids to about 210 aminoacids, about 20 amino acids to about 205 amino acids, about 20 aminoacids to about 200 amino acids, about 20 amino acids to about 195 aminoacids, about 20 amino acids to about 190 amino acids, about 20 aminoacids to about 185 amino acids, about 20 amino acids to about 180 aminoacids, about 20 amino acids to about 175 amino acids, about 20 aminoacids to about 170 amino acids, about 20 amino acids to about 165 aminoacids, about 20 amino acids to about 160 amino acids, about 20 aminoacids to about 155 amino acids, about 20 amino acids to about 150 aminoacids, about 20 amino acids to about 145 amino acids, about 20 aminoacids to about 140 amino acids, about 20 amino acids to about 135 aminoacids, about 20 amino acids to about 130 amino acids, about 20 aminoacids to about 125 amino acids, about 20 amino acids to about 120 aminoacids, about 20 amino acids to about 115 amino acids, about 20 aminoacids to about 110 amino acids, about 20 amino acids to about 105 aminoacids, about 20 amino acids to about 100 amino acids, about 20 aminoacids to about 95 amino acids, about 20 amino acids to about 90 aminoacids, about 20 amino acids to about 85 amino acids, about 20 aminoacids to about 80 amino acids, about 20 amino acids to about 75 aminoacids, about 20 amino acids to about 70 amino acids, about 20 aminoacids to about 60 amino acids, about 20 amino acids to about 50 aminoacids, about 20 amino acids to about 40 amino acids, about 20 aminoacids to about 30 amino acids, about 30 amino acids to about 220 aminoacids, about 30 amino acids to about 215 amino acids, about 30 aminoacids to about 210 amino acids, about 30 amino acids to about 205 aminoacids, about 30 amino acids to about 200 amino acids, about 30 aminoacids to about 195 amino acids, about 30 amino acids to about 190 aminoacids, about 30 amino acids to about 185 amino acids, about 30 aminoacids to about 180 amino acids, about 30 amino acids to about 175 aminoacids, about 30 amino acids to about 170 amino acids, about 30 aminoacids to about 165 amino acids, about 30 amino acids to about 160 aminoacids, about 30 amino acids to about 155 amino acids, about 30 aminoacids to about 150 amino acids, about 30 amino acids to about 145 aminoacids, about 30 amino acids to about 140 amino acids, about 30 aminoacids to about 135 amino acids, about 30 amino acids to about 130 aminoacids, about 30 amino acids to about 125 amino acids, about 30 aminoacids to about 120 amino acids, about 30 amino acids to about 115 aminoacids, about 30 amino acids to about 110 amino acids, about 30 aminoacids to about 105 amino acids, about 30 amino acids to about 100 aminoacids, about 30 amino acids to about 95 amino acids, about 30 aminoacids to about 90 amino acids, about 30 amino acids to about 85 aminoacids, about 30 amino acids to about 80 amino acids, about 30 aminoacids to about 75 amino acids, about 30 amino acids to about 70 aminoacids, about 30 amino acids to about 60 amino acids, about 30 aminoacids to about 50 amino acids, about 30 amino acids to about 40 aminoacids, about 40 amino acids to about 220 amino acids, about 40 aminoacids to about 215 amino acids, about 40 amino acids to about 210 aminoacids, about 40 amino acids to about 205 amino acids, about 40 aminoacids to about 200 amino acids, about 40 amino acids to about 195 aminoacids, about 40 amino acids to about 190 amino acids, about 40 aminoacids to about 185 amino acids, about 40 amino acids to about 180 aminoacids, about 40 amino acids to about 175 amino acids, about 40 aminoacids to about 170 amino acids, about 40 amino acids to about 165 aminoacids, about 40 amino acids to about 160 amino acids, about 40 aminoacids to about 155 amino acids, about 40 amino acids to about 150 aminoacids, about 40 amino acids to about 145 amino acids, about 40 aminoacids to about 140 amino acids, about 40 amino acids to about 135 aminoacids, about 40 amino acids to about 130 amino acids, about 40 aminoacids to about 125 amino acids, about 40 amino acids to about 120 aminoacids, about 40 amino acids to about 115 amino acids, about 40 aminoacids to about 110 amino acids, about 40 amino acids to about 105 aminoacids, about 40 amino acids to about 100 amino acids, about 40 aminoacids to about 95 amino acids, about 40 amino acids to about 90 aminoacids, about 40 amino acids to about 85 amino acids, about 40 aminoacids to about 80 amino acids, about 40 amino acids to about 75 aminoacids, about 40 amino acids to about 70 amino acids, about 40 aminoacids to about 60 amino acids, about 40 amino acids to about 50 aminoacids, about 50 amino acids to about 220 amino acids, about 50 aminoacids to about 215 amino acids, about 50 amino acids to about 210 aminoacids, about 50 amino acids to about 205 amino acids, about 50 aminoacids to about 200 amino acids, about 50 amino acids to about 195 aminoacids, about 50 amino acids to about 190 amino acids, about 50 aminoacids to about 185 amino acids, about 50 amino acids to about 180 aminoacids, about 50 amino acids to about 175 amino acids, about 50 aminoacids to about 170 amino acids, about 50 amino acids to about 165 aminoacids, about 50 amino acids to about 160 amino acids, about 50 aminoacids to about 155 amino acids, about 50 amino acids to about 150 aminoacids, about 50 amino acids to about 145 amino acids, about 50 aminoacids to about 140 amino acids, about 50 amino acids to about 135 aminoacids, about 50 amino acids to about 130 amino acids, about 50 aminoacids to about 125 amino acids, about 50 amino acids to about 120 aminoacids, about 50 amino acids to about 115 amino acids, about 50 aminoacids to about 110 amino acids, about 50 amino acids to about 105 aminoacids, about 50 amino acids to about 100 amino acids, about 50 aminoacids to about 95 amino acids, about 50 amino acids to about 90 aminoacids, about 50 amino acids to about 85 amino acids, about 50 aminoacids to about 80 amino acids, about 50 amino acids to about 75 aminoacids, about 50 amino acids to about 70 amino acids, about 50 aminoacids to about 60 amino acids, about 60 amino acids to about 220 aminoacids, about 60 amino acids to about 215 amino acids, about 60 aminoacids to about 210 amino acids, about 60 amino acids to about 205 aminoacids, about 60 amino acids to about 200 amino acids, about 60 aminoacids to about 195 amino acids, about 60 amino acids to about 190 aminoacids, about 60 amino acids to about 185 amino acids, about 60 aminoacids to about 180 amino acids, about 60 amino acids to about 175 aminoacids, about 60 amino acids to about 170 amino acids, about 60 aminoacids to about 165 amino acids, about 60 amino acids to about 160 aminoacids, about 60 amino acids to about 155 amino acids, about 60 aminoacids to about 150 amino acids, about 60 amino acids to about 145 aminoacids, about 60 amino acids to about 140 amino acids, about 60 aminoacids to about 135 amino acids, about 60 amino acids to about 130 aminoacids, about 60 amino acids to about 125 amino acids, about 60 aminoacids to about 120 amino acids, about 60 amino acids to about 115 aminoacids, about 60 amino acids to about 110 amino acids, about 60 aminoacids to about 105 amino acids, about 60 amino acids to about 100 aminoacids, about 60 amino acids to about 95 amino acids, about 60 aminoacids to about 90 amino acids, about 60 amino acids to about 85 aminoacids, about 60 amino acids to about 80 amino acids, about 60 aminoacids to about 75 amino acids, about 60 amino acids to about 70 aminoacids, about 70 amino acids to about 220 amino acids, about 70 aminoacids to about 215 amino acids, about 70 amino acids to about 210 aminoacids, about 70 amino acids to about 205 amino acids, about 70 aminoacids to about 200 amino acids, about 70 amino acids to about 195 aminoacids, about 70 amino acids to about 190 amino acids, about 70 aminoacids to about 185 amino acids, about 70 amino acids to about 180 aminoacids, about 70 amino acids to about 175 amino acids, about 70 aminoacids to about 170 amino acids, about 70 amino acids to about 165 aminoacids, about 70 amino acids to about 160 amino acids, about 70 aminoacids to about 155 amino acids, about 70 amino acids to about 150 aminoacids, about 70 amino acids to about 145 amino acids, about 70 aminoacids to about 140 amino acids, about 70 amino acids to about 135 aminoacids, about 70 amino acids to about 130 amino acids, about 70 aminoacids to about 125 amino acids, about 70 amino acids to about 120 aminoacids, about 70 amino acids to about 115 amino acids, about 70 aminoacids to about 110 amino acids, about 70 amino acids to about 105 aminoacids, about 70 amino acids to about 100 amino acids, about 70 aminoacids to about 95 amino acids, about 70 amino acids to about 90 aminoacids, about 70 amino acids to about 85 amino acids, about 70 aminoacids to about 80 amino acids, about 80 amino acids to about 220 aminoacids, about 80 amino acids to about 215 amino acids, about 80 aminoacids to about 210 amino acids, about 80 amino acids to about 205 aminoacids, about 80 amino acids to about 200 amino acids, about 80 aminoacids to about 195 amino acids, about 80 amino acids to about 190 aminoacids, about 80 amino acids to about 185 amino acids, about 80 aminoacids to about 180 amino acids, about 80 amino acids to about 175 aminoacids, about 80 amino acids to about 170 amino acids, about 80 aminoacids to about 165 amino acids, about 80 amino acids to about 160 aminoacids, about 80 amino acids to about 155 amino acids, about 80 aminoacids to about 150 amino acids, about 80 amino acids to about 145 aminoacids, about 80 amino acids to about 140 amino acids, about 80 aminoacids to about 135 amino acids, about 80 amino acids to about 130 aminoacids, about 80 amino acids to about 125 amino acids, about 80 aminoacids to about 120 amino acids, about 80 amino acids to about 115 aminoacids, about 80 amino acids to about 110 amino acids, about 80 aminoacids to about 105 amino acids, about 80 amino acids to about 100 aminoacids, about 80 amino acids to about 95 amino acids, about 80 aminoacids to about 90 amino acids, about 90 amino acids to about 220 aminoacids, about 90 amino acids to about 215 amino acids, about 90 aminoacids to about 210 amino acids, about 90 amino acids to about 205 aminoacids, about 90 amino acids to about 200 amino acids, about 90 aminoacids to about 195 amino acids, about 90 amino acids to about 190 aminoacids, about 90 amino acids to about 185 amino acids, about 90 aminoacids to about 180 amino acids, about 90 amino acids to about 175 aminoacids, about 90 amino acids to about 170 amino acids, about 90 aminoacids to about 165 amino acids, about 90 amino acids to about 160 aminoacids, about 90 amino acids to about 155 amino acids, about 90 aminoacids to about 150 amino acids, about 90 amino acids to about 145 aminoacids, about 90 amino acids to about 140 amino acids, about 90 aminoacids to about 135 amino acids, about 90 amino acids to about 130 aminoacids, about 90 amino acids to about 125 amino acids, about 90 aminoacids to about 120 amino acids, about 90 amino acids to about 115 aminoacids, about 90 amino acids to about 110 amino acids, about 90 aminoacids to about 105 amino acids, about 90 amino acids to about 100 aminoacids, about 100 amino acids to about 220 amino acids, about 100 aminoacids to about 215 amino acids, about 100 amino acids to about 210 aminoacids, about 100 amino acids to about 205 amino acids, about 100 aminoacids to about 200 amino acids, about 100 amino acids to about 195 aminoacids, about 100 amino acids to about 190 amino acids, about 100 aminoacids to about 185 amino acids, about 100 amino acids to about 180 aminoacids, about 100 amino acids to about 175 amino acids, about 100 aminoacids to about 170 amino acids, about 100 amino acids to about 165 aminoacids, about 100 amino acids to about 160 amino acids, about 100 aminoacids to about 155 amino acids, about 100 amino acids to about 150 aminoacids, about 100 amino acids to about 145 amino acids, about 100 aminoacids to about 140 amino acids, about 100 amino acids to about 135 aminoacids, about 100 amino acids to about 130 amino acids, about 100 aminoacids to about 125 amino acids, about 100 amino acids to about 120 aminoacids, about 100 amino acids to about 115 amino acids, about 100 aminoacids to about 110 amino acids, about 110 amino acids to about 220 aminoacids, about 110 amino acids to about 215 amino acids, about 110 aminoacids to about 210 amino acids, about 110 amino acids to about 205 aminoacids, about 110 amino acids to about 200 amino acids, about 110 aminoacids to about 195 amino acids, about 110 amino acids to about 190 aminoacids, about 110 amino acids to about 185 amino acids, about 110 aminoacids to about 180 amino acids, about 110 amino acids to about 175 aminoacids, about 110 amino acids to about 170 amino acids, about 110 aminoacids to about 165 amino acids, about 110 amino acids to about 160 aminoacids, about 110 amino acids to about 155 amino acids, about 110 aminoacids to about 150 amino acids, about 110 amino acids to about 145 aminoacids, about 110 amino acids to about 140 amino acids, about 110 aminoacids to about 135 amino acids, about 110 amino acids to about 130 aminoacids, about 110 amino acids to about 125 amino acids, about 110 aminoacids to about 120 amino acids, about 110 amino acids to about 115 aminoacids, about 115 amino acids to about 220 amino acids, about 115 aminoacids to about 215 amino acids, about 115 amino acids to about 210 aminoacids, about 115 amino acids to about 205 amino acids, about 115 aminoacids to about 200 amino acids, about 115 amino acids to about 195 aminoacids, about 115 amino acids to about 190 amino acids, about 115 aminoacids to about 185 amino acids, about 115 amino acids to about 180 aminoacids, about 115 amino acids to about 175 amino acids, about 115 aminoacids to about 170 amino acids, about 115 amino acids to about 165 aminoacids, about 115 amino acids to about 160 amino acids, about 115 aminoacids to about 155 amino acids, about 115 amino acids to about 150 aminoacids, about 115 amino acids to about 145 amino acids, about 115 aminoacids to about 140 amino acids, about 115 amino acids to about 135 aminoacids, about 115 amino acids to about 130 amino acids, about 115 aminoacids to about 125 amino acids, about 115 amino acids to about 120 aminoacids, about 120 amino acids to about 220 amino acids, about 120 aminoacids to about 215 amino acids, about 120 amino acids to about 210 aminoacids, about 120 amino acids to about 205 amino acids, about 120 aminoacids to about 200 amino acids, about 120 amino acids to about 195 aminoacids, about 120 amino acids to about 190 amino acids, about 120 aminoacids to about 185 amino acids, about 120 amino acids to about 180 aminoacids, about 120 amino acids to about 175 amino acids, about 120 aminoacids to about 170 amino acids, about 120 amino acids to about 165 aminoacids, about 120 amino acids to about 160 amino acids, about 120 aminoacids to about 155 amino acids, about 120 amino acids to about 150 aminoacids, about 120 amino acids to about 145 amino acids, about 120 aminoacids to about 140 amino acids, about 120 amino acids to about 135 aminoacids, about 120 amino acids to about 130 amino acids, about 120 aminoacids to about 125 amino acids, about 125 amino acids to about 220 aminoacids, about 125 amino acids to about 215 amino acids, about 125 aminoacids to about 210 amino acids, about 125 amino acids to about 205 aminoacids, about 125 amino acids to about 200 amino acids, about 125 aminoacids to about 195 amino acids, about 125 amino acids to about 190 aminoacids, about 125 amino acids to about 185 amino acids, about 125 aminoacids to about 180 amino acids, about 125 amino acids to about 175 aminoacids, about 125 amino acids to about 170 amino acids, about 125 aminoacids to about 165 amino acids, about 125 amino acids to about 160 aminoacids, about 125 amino acids to about 155 amino acids, about 125 aminoacids to about 150 amino acids, about 125 amino acids to about 145 aminoacids, about 125 amino acids to about 140 amino acids, about 125 aminoacids to about 135 amino acids, about 125 amino acids to about 130 aminoacids, about 130 amino acids to about 220 amino acids, about 130 aminoacids to about 215 amino acids, about 130 amino acids to about 210 aminoacids, about 130 amino acids to about 205 amino acids, about 130 aminoacids to about 200 amino acids, about 130 amino acids to about 195 aminoacids, about 130 amino acids to about 190 amino acids, about 130 aminoacids to about 185 amino acids, about 130 amino acids to about 180 aminoacids, about 130 amino acids to about 175 amino acids, about 130 aminoacids to about 170 amino acids, about 130 amino acids to about 165 aminoacids, about 130 amino acids to about 160 amino acids, about 130 aminoacids to about 155 amino acids, about 130 amino acids to about 150 aminoacids, about 130 amino acids to about 145 amino acids, about 130 aminoacids to about 140 amino acids, about 130 amino acids to about 135 aminoacids, about 135 amino acids to about 220 amino acids, about 135 aminoacids to about 215 amino acids, about 135 amino acids to about 210 aminoacids, about 135 amino acids to about 205 amino acids, about 135 aminoacids to about 200 amino acids, about 135 amino acids to about 195 aminoacids, about 135 amino acids to about 190 amino acids, about 135 aminoacids to about 185 amino acids, about 135 amino acids to about 180 aminoacids, about 135 amino acids to about 175 amino acids, about 135 aminoacids to about 170 amino acids, about 135 amino acids to about 165 aminoacids, about 135 amino acids to about 160 amino acids, about 135 aminoacids to about 155 amino acids, about 135 amino acids to about 150 aminoacids, about 135 amino acids to about 145 amino acids, about 135 aminoacids to about 140 amino acids, about 140 amino acids to about 220 aminoacids, about 140 amino acids to about 215 amino acids, about 140 aminoacids to about 210 amino acids, about 140 amino acids to about 205 aminoacids, about 140 amino acids to about 200 amino acids, about 140 aminoacids to about 195 amino acids, about 140 amino acids to about 190 aminoacids, about 140 amino acids to about 185 amino acids, about 140 aminoacids to about 180 amino acids, about 140 amino acids to about 175 aminoacids, about 140 amino acids to about 170 amino acids, about 140 aminoacids to about 165 amino acids, about 140 amino acids to about 160 aminoacids, about 140 amino acids to about 155 amino acids, about 140 aminoacids to about 150 amino acids, about 140 amino acids to about 145 aminoacids, about 145 amino acids to about 220 amino acids, about 145 aminoacids to about 215 amino acids, about 145 amino acids to about 210 aminoacids, about 145 amino acids to about 205 amino acids, about 145 aminoacids to about 200 amino acids, about 145 amino acids to about 195 aminoacids, about 145 amino acids to about 190 amino acids, about 145 aminoacids to about 185 amino acids, about 145 amino acids to about 180 aminoacids, about 145 amino acids to about 175 amino acids, about 145 aminoacids to about 170 amino acids, about 145 amino acids to about 165 aminoacids, about 145 amino acids to about 160 amino acids, about 145 aminoacids to about 155 amino acids, about 145 amino acids to about 150 aminoacids, about 150 amino acids to about 220 amino acids, about 150 aminoacids to about 215 amino acids, about 150 amino acids to about 210 aminoacids, about 150 amino acids to about 205 amino acids, about 150 aminoacids to about 200 amino acids, about 150 amino acids to about 195 aminoacids, about 150 amino acids to about 190 amino acids, about 150 aminoacids to about 185 amino acids, about 150 amino acids to about 180 aminoacids, about 150 amino acids to about 175 amino acids, about 150 aminoacids to about 170 amino acids, about 150 amino acids to about 165 aminoacids, about 150 amino acids to about 160 amino acids, about 150 aminoacids to about 155 amino acids, about 155 amino acids to about 220 aminoacids, about 155 amino acids to about 215 amino acids, about 155 aminoacids to about 210 amino acids, about 155 amino acids to about 205 aminoacids, about 155 amino acids to about 200 amino acids, about 155 aminoacids to about 195 amino acids, about 155 amino acids to about 190 aminoacids, about 155 amino acids to about 185 amino acids, about 155 aminoacids to about 180 amino acids, about 155 amino acids to about 175 aminoacids, about 155 amino acids to about 170 amino acids, about 155 aminoacids to about 165 amino acids, about 155 amino acids to about 160 aminoacids, about 160 amino acids to about 220 amino acids, about 160 aminoacids to about 215 amino acids, about 160 amino acids to about 210 aminoacids, about 160 amino acids to about 205 amino acids, about 160 aminoacids to about 200 amino acids, about 160 amino acids to about 195 aminoacids, about 160 amino acids to about 190 amino acids, about 160 aminoacids to about 185 amino acids, about 160 amino acids to about 180 aminoacids, about 160 amino acids to about 175 amino acids, about 160 aminoacids to about 170 amino acids, about 160 amino acids to about 165 aminoacids, about 165 amino acids to about 220 amino acids, about 165 aminoacids to about 215 amino acids, about 165 amino acids to about 210 aminoacids, about 165 amino acids to about 205 amino acids, about 165 aminoacids to about 200 amino acids, about 165 amino acids to about 195 aminoacids, about 165 amino acids to about 190 amino acids, about 165 aminoacids to about 185 amino acids, about 165 amino acids to about 180 aminoacids, about 165 amino acids to about 175 amino acids, about 165 aminoacids to about 170 amino acids, about 170 amino acids to about 220 aminoacids, about 170 amino acids to about 215 amino acids, about 170 aminoacids to about 210 amino acids, about 170 amino acids to about 205 aminoacids, about 170 amino acids to about 200 amino acids, about 170 aminoacids to about 195 amino acids, about 170 amino acids to about 190 aminoacids, about 170 amino acids to about 185 amino acids, about 170 aminoacids to about 180 amino acids, about 170 amino acids to about 175 aminoacids, about 175 amino acids to about 220 amino acids, about 175 aminoacids to about 215 amino acids, about 175 amino acids to about 210 aminoacids, about 175 amino acids to about 205 amino acids, about 175 aminoacids to about 200 amino acids, about 175 amino acids to about 195 aminoacids, about 175 amino acids to about 190 amino acids, about 175 aminoacids to about 185 amino acids, about 175 amino acids to about 180 aminoacids, about 180 amino acids to about 220 amino acids, about 180 aminoacids to about 215 amino acids, about 180 amino acids to about 210 aminoacids, about 180 amino acids to about 205 amino acids, about 180 aminoacids to about 200 amino acids, about 180 amino acids to about 195 aminoacids, about 180 amino acids to about 190 amino acids, about 180 aminoacids to about 185 amino acids, about 185 amino acids to about 220 aminoacids, about 185 amino acids to about 215 amino acids, about 185 aminoacids to about 210 amino acids, about 185 amino acids to about 205 aminoacids, about 185 amino acids to about 200 amino acids, about 185 aminoacids to about 195 amino acids, about 185 amino acids to about 190 aminoacids, about 190 amino acids to about 220 amino acids, about 190 aminoacids to about 215 amino acids, about 190 amino acids to about 210 aminoacids, about 190 amino acids to about 205 amino acids, about 190 aminoacids to about 200 amino acids, about 190 amino acids to about 195 aminoacids, about 195 amino acids to about 220 amino acids, about 195 aminoacids to about 215 amino acids, about 195 amino acids to about 210 aminoacids, about 195 amino acids to about 205 amino acids, about 195 aminoacids to about 200 amino acids, about 200 amino acids to about 220 aminoacids, about 200 amino acids to about 215 amino acids, about 200 aminoacids to about 210 amino acids, about 200 amino acids to about 205 aminoacids, about 205 amino acids to about 220 amino acids, about 205 aminoacids to about 215 amino acids, about 205 amino acids to about 210 aminoacids, about 210 amino acids to about 220 amino acids, about 210 aminoacids to about 215 amino acids, or about 215 amino acids to about 220amino acids.

Linker Sequences

In some embodiments, the linker sequence can be a flexible linkersequence. Non-limiting examples of linker sequences that can be used aredescribed in Klein et al., Protein Engineering, Design & Selection27(10):325-330, 2014; Priyanka et al., Protein Sci. 22(2):153-167, 2013.In some examples, the linker sequence is a synthetic linker sequence.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first chimeric polypeptide can include one, two,three, four, five, six, seven, eight, nine, or ten linker sequence(s)(e.g., the same or different linker sequences, e.g., any of theexemplary linker sequences described herein or known in the art). Insome embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the second chimeric polypeptide can include one, two,three, four, five, six, seven, eight, nine, or ten linker sequence(s)(e.g., the same or different linker sequences, e.g., any of theexemplary linker sequences described herein or known in the art).

In some embodiments, a linker sequence can have a total length of 1amino acid to about 100 amino acids, 1 amino acid to about 90 aminoacids, 1 amino acid to about 80 amino acids, 1 amino acid to about 70amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid toabout 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acidto about 30 amino acids, 1 amino acid to about 25 amino acids, 1 aminoacid to about 24 amino acids, 1 amino acid to about 22 amino acids, 1amino acid to about 20 amino acids, 1 amino acid to about 18 aminoacids, 1 amino acid to about 16 amino acids, 1 amino acid to about 14amino acids, 1 amino acid to about 12 amino acids, 1 amino acid to about10 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid toabout 6 amino acids, 1 amino acid to about 4 amino acids, about 2 aminoacids to about 100 amino acids, about 2 amino acids to about 90 aminoacids, about 2 amino acids to about 80 amino acids, about 2 amino acidsto about 70 amino acids, about 2 amino acids to about 60 amino acids,about 2 amino acids to about 50 amino acids, about 2 amino acids toabout 45 amino acids, about 2 amino acids to about 40 amino acids, about2 amino acids to about 35 amino acids, about 2 amino acids to about 30amino acids, about 2 amino acids to about 25 amino acids, about 2 aminoacids to about 24 amino acids, about 2 amino acids to about 22 aminoacids, about 2 amino acids to about 20 amino acids, about 2 amino acidsto about 18 amino acids, about 2 amino acids to about 16 amino acids,about 2 amino acids to about 14 amino acids, about 2 amino acids toabout 12 amino acids, about 2 amino acids to about 10 amino acids, about2 amino acids to about 8 amino acids, about 2 amino acids to about 6amino acids, about 2 amino acids to about 4 amino acids, about 4 aminoacids to about 100 amino acids, about 4 amino acids to about 90 aminoacids, about 4 amino acids to about 80 amino acids, about 4 amino acidsto about 70 amino acids, about 4 amino acids to about 60 amino acids,about 4 amino acids to about 50 amino acids, about 4 amino acids toabout 45 amino acids, about 4 amino acids to about 40 amino acids, about4 amino acids to about 35 amino acids, about 4 amino acids to about 30amino acids, about 4 amino acids to about 25 amino acids, about 4 aminoacids to about 24 amino acids, about 4 amino acids to about 22 aminoacids, about 4 amino acids to about 20 amino acids, about 4 amino acidsto about 18 amino acids, about 4 amino acids to about 16 amino acids,about 4 amino acids to about 14 amino acids, about 4 amino acids toabout 12 amino acids, about 4 amino acids to about 10 amino acids, about4 amino acids to about 8 amino acids, about 4 amino acids to about 6amino acids, about 6 amino acids to about 100 amino acids, about 6 aminoacids to about 90 amino acids, about 6 amino acids to about 80 aminoacids, about 6 amino acids to about 70 amino acids, about 6 amino acidsto about 60 amino acids, about 6 amino acids to about 50 amino acids,about 6 amino acids to about 45 amino acids, about 6 amino acids toabout 40 amino acids, about 6 amino acids to about 35 amino acids, about6 amino acids to about 30 amino acids, about 6 amino acids to about 25amino acids, about 6 amino acids to about 24 amino acids, about 6 aminoacids to about 22 amino acids, about 6 amino acids to about 20 aminoacids, about 6 amino acids to about 18 amino acids, about 6 amino acidsto about 16 amino acids, about 6 amino acids to about 14 amino acids,about 6 amino acids to about 12 amino acids, about 6 amino acids toabout 10 amino acids, about 6 amino acids to about 8 amino acids, about8 amino acids to about 100 amino acids, about 8 amino acids to about 90amino acids, about 8 amino acids to about 80 amino acids, about 8 aminoacids to about 70 amino acids, about 8 amino acids to about 60 aminoacids, about 8 amino acids to about 50 amino acids, about 8 amino acidsto about 45 amino acids, about 8 amino acids to about 40 amino acids,about 8 amino acids to about 35 amino acids, about 8 amino acids toabout 30 amino acids, about 8 amino acids to about 25 amino acids, about8 amino acids to about 24 amino acids, about 8 amino acids to about 22amino acids, about 8 amino acids to about 20 amino acids, about 8 aminoacids to about 18 amino acids, about 8 amino acids to about 16 aminoacids, about 8 amino acids to about 14 amino acids, about 8 amino acidsto about 12 amino acids, about 8 amino acids to about 10 amino acids,about 10 amino acids to about 100 amino acids, about 10 amino acids toabout 90 amino acids, about 10 amino acids to about 80 amino acids,about 10 amino acids to about 70 amino acids, about 10 amino acids toabout 60 amino acids, about 10 amino acids to about 50 amino acids,about 10 amino acids to about 45 amino acids, about 10 amino acids toabout 40 amino acids, about 10 amino acids to about 35 amino acids,about 10 amino acids to about 30 amino acids, about 10 amino acids toabout 25 amino acids, about 10 amino acids to about 24 amino acids,about 10 amino acids to about 22 amino acids, about 10 amino acids toabout 20 amino acids, about 10 amino acids to about 18 amino acids,about 10 amino acids to about 16 amino acids, about 10 amino acids toabout 14 amino acids, about 10 amino acids to about 12 amino acids,about 12 amino acids to about 100 amino acids, about 12 amino acids toabout 90 amino acids, about 12 amino acids to about 80 amino acids,about 12 amino acids to about 70 amino acids, about 12 amino acids toabout 60 amino acids, about 12 amino acids to about 50 amino acids,about 12 amino acids to about 45 amino acids, about 12 amino acids toabout 40 amino acids, about 12 amino acids to about 35 amino acids,about 12 amino acids to about 30 amino acids, about 12 amino acids toabout 25 amino acids, about 12 amino acids to about 24 amino acids,about 12 amino acids to about 22 amino acids, about 12 amino acids toabout 20 amino acids, about 12 amino acids to about 18 amino acids,about 12 amino acids to about 16 amino acids, about 12 amino acids toabout 14 amino acids, about 14 amino acids to about 100 amino acids,about 14 amino acids to about 90 amino acids, about 14 amino acids toabout 80 amino acids, about 14 amino acids to about 70 amino acids,about 14 amino acids to about 60 amino acids, about 14 amino acids toabout 50 amino acids, about 14 amino acids to about 45 amino acids,about 14 amino acids to about 40 amino acids, about 14 amino acids toabout 35 amino acids, about 14 amino acids to about 30 amino acids,about 14 amino acids to about 25 amino acids, about 14 amino acids toabout 24 amino acids, about 14 amino acids to about 22 amino acids,about 14 amino acids to about 20 amino acids, about 14 amino acids toabout 18 amino acids, about 14 amino acids to about 16 amino acids,about 16 amino acids to about 100 amino acids, about 16 amino acids toabout 90 amino acids, about 16 amino acids to about 80 amino acids,about 16 amino acids to about 70 amino acids, about 16 amino acids toabout 60 amino acids, about 16 amino acids to about 50 amino acids,about 16 amino acids to about 45 amino acids, about 16 amino acids toabout 40 amino acids, about 16 amino acids to about 35 amino acids,about 16 amino acids to about 30 amino acids, about 16 amino acids toabout 25 amino acids, about 16 amino acids to about 24 amino acids,about 16 amino acids to about 22 amino acids, about 16 amino acids toabout 20 amino acids, about 16 amino acids to about 18 amino acids,about 18 amino acids to about 100 amino acids, about 18 amino acids toabout 90 amino acids, about 18 amino acids to about 80 amino acids,about 18 amino acids to about 70 amino acids, about 18 amino acids toabout 60 amino acids, about 18 amino acids to about 50 amino acids,about 18 amino acids to about 45 amino acids, about 18 amino acids toabout 40 amino acids, about 18 amino acids to about 35 amino acids,about 18 amino acids to about 30 amino acids, about 18 amino acids toabout 25 amino acids, about 18 amino acids to about 24 amino acids,about 18 amino acids to about 22 amino acids, about 18 amino acids toabout 20 amino acids, about 20 amino acids to about 100 amino acids,about 20 amino acids to about 90 amino acids, about 20 amino acids toabout 80 amino acids, about 20 amino acids to about 70 amino acids,about 20 amino acids to about 60 amino acids, about 20 amino acids toabout 50 amino acids, about 20 amino acids to about 45 amino acids,about 20 amino acids to about 40 amino acids, about 20 amino acids toabout 35 amino acids, about 20 amino acids to about 30 amino acids,about 20 amino acids to about 25 amino acids, about 20 amino acids toabout 24 amino acids, about 20 amino acids to about 22 amino acids,about 22 amino acids to about 100 amino acids, about 22 amino acids toabout 90 amino acids, about 22 amino acids to about 80 amino acids,about 22 amino acids to about 70 amino acids, about 22 amino acids toabout 60 amino acids, about 22 amino acids to about 50 amino acids,about 22 amino acids to about 45 amino acids, about 22 amino acids toabout 40 amino acids, about 22 amino acids to about 35 amino acids,about 22 amino acids to about 30 amino acids, about 22 amino acids toabout 25 amino acids, about 22 amino acids to about 24 amino acids,about 25 amino acids to about 100 amino acids, about 25 amino acids toabout 90 amino acids, about 25 amino acids to about 80 amino acids,about 25 amino acids to about 70 amino acids, about 25 amino acids toabout 60 amino acids, about 25 amino acids to about 50 amino acids,about 25 amino acids to about 45 amino acids, about 25 amino acids toabout 40 amino acids, about 25 amino acids to about 35 amino acids,about 25 amino acids to about 30 amino acids, about 30 amino acids toabout 100 amino acids, about 30 amino acids to about 90 amino acids,about 30 amino acids to about 80 amino acids, about 30 amino acids toabout 70 amino acids, about 30 amino acids to about 60 amino acids,about 30 amino acids to about 50 amino acids, about 30 amino acids toabout 45 amino acids, about 30 amino acids to about 40 amino acids,about 30 amino acids to about 35 amino acids, about 35 amino acids toabout 100 amino acids, about 35 amino acids to about 90 amino acids,about 35 amino acids to about 80 amino acids, about 35 amino acids toabout 70 amino acids, about 35 amino acids to about 60 amino acids,about 35 amino acids to about 50 amino acids, about 35 amino acids toabout 45 amino acids, about 35 amino acids to about 40 amino acids,about 40 amino acids to about 100 amino acids, about 40 amino acids toabout 90 amino acids, about 40 amino acids to about 80 amino acids,about 40 amino acids to about 70 amino acids, about 40 amino acids toabout 60 amino acids, about 40 amino acids to about 50 amino acids,about 40 amino acids to about 45 amino acids, about 45 amino acids toabout 100 amino acids, about 45 amino acids to about 90 amino acids,about 45 amino acids to about 80 amino acids, about 45 amino acids toabout 70 amino acids, about 45 amino acids to about 60 amino acids,about 45 amino acids to about 50 amino acids, about 50 amino acids toabout 100 amino acids, about 50 amino acids to about 90 amino acids,about 50 amino acids to about 80 amino acids, about 50 amino acids toabout 70 amino acids, about 50 amino acids to about 60 amino acids,about 60 amino acids to about 100 amino acids, about 60 amino acids toabout 90 amino acids, about 60 amino acids to about 80 amino acids,about 60 amino acids to about 70 amino acids, about 70 amino acids toabout 100 amino acids, about 70 amino acids to about 90 amino acids,about 70 amino acids to about 80 amino acids, about 80 amino acids toabout 100 amino acids, about 80 amino acids to about 90 amino acids, orabout 90 amino acids to about 100 amino acids.

In some embodiments, the linker is rich in glycine (Gly or G) residues.In some embodiments, the linker is rich in serine (Ser or S) residues.In some embodiments, the linker is rich in glycine and serine residues.In some embodiments, the linker has one or more glycine-serine residuepairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. Insome embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS)sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGSsequences. In some embodiments, the linker has one or moreGly-Gly-Gly-Gly-Ser (GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 or more GGGGS sequences. In some embodiments, the linker has oneor more Gly-Gly-Ser-Gly (GGSG) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more GGSG sequences.

In some embodiments, the linker sequence can comprise or consist ofGGGGSGGGGSGGGGS (SEQ ID NO: 7). In some embodiments, the linker sequencecan be encoded by a nucleic acid comprising or consisting of:GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 8). In someembodiments, the linker sequence can comprise or consist of:

(SEQ ID NO: 9) GGGSGGGS.Target-Binding Domains

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain, the secondtarget-binding domain, and/or the additional one or more target-bindingdomains can be an antigen-binding domain (e.g., any of the exemplaryantigen-binding domains described herein or known in the art), a solubleinterleukin or cytokine protein (e.g., any of the exemplary solubleinterleukin proteins or soluble cytokine proteins described herein), anda soluble interleukin or cytokine receptor (e.g., any of the exemplarysoluble interleukin receptors or soluble cytokine receptors describedherein).

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, one or more of the first target-binding domain (e.g.,any of the exemplary first target binding domains described herein orknown in the art), the second target-binding domain (e.g., any of theexemplary second target binding domains described herein or known in theart), and the one or more additional target binding domains can each,independently, bind specifically to a target selected from the group of:bind specifically to a target selected from the group consisting of:CD16a, CD28, CD3 (e.g., one or more of CD3α, CD3β, CD3δ, CD3ε, andCD3γ), CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4,IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα,CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET,EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein (e.g., ULBP1, ULBP2, ULBP3, ULBP4,ULBP5, and ULBP6), HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD,a ligand of TGF-β receptor II (TGF-β RII), a ligand of TGF-β RIII, aligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand ofNKG2D, a ligand of NK_(P)30, a ligand for a scMHCI, a ligand for ascMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor for IL-2,a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, a receptor for CD122, and a receptor for CD28.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain, the secondtarget-binding domain, and/or the one or more additional target-bindingdomains can each independent have a total number of amino acids of about5 amino acids to about 1000 amino acids, about 5 amino acids to about950 amino acids, about 5 amino acids to about 900 amino acids, about 5amino acids to about 850 amino acids, about 5 amino acids to about 800amino acids, about 5 amino acids to about 750 amino acids, about 5 aminoacids to about 700 amino acids, about 5 amino acids to about 650 aminoacids, about 5 amino acids to about 600 amino acids, about 5 amino acidsto about 550 amino acids, about 5 amino acids to about 500 amino acids,about 5 amino acids to about 450 amino acids, about 5 amino acids toabout 400 amino acids, about 5 amino acids to about 350 amino acids,about 5 amino acids to about 300 amino acids, about 5 amino acids toabout 280 amino acids, about 5 amino acids to about 260 amino acids,about 5 amino acids to about 240 amino acids, about 5 amino acids toabout 220 amino acids, about 5 amino acids to about 200 amino acids,about 5 amino acids to about 195 amino acids, about 5 amino acids toabout 190 amino acids, about 5 amino acids to about 185 amino acids,about 5 amino acids to about 180 amino acids, about 5 amino acids toabout 175 amino acids, about 5 amino acids to about 170 amino acids,about 5 amino acids to about 165 amino acids, about 5 amino acids toabout 160 amino acids, about 5 amino acids to about 155 amino acids,about 5 amino acids to about 150 amino acids, about 5 amino acids toabout 145 amino acids, about 5 amino acids to about 140 amino acids,about 5 amino acids to about 135 amino acids, about 5 amino acids toabout 130 amino acids, about 5 amino acids to about 125 amino acids,about 5 amino acids to about 120 amino acids, about 5 amino acids toabout 115 amino acids, about 5 amino acids to about 110 amino acids,about 5 amino acids to about 105 amino acids, about 5 amino acids toabout 100 amino acids, about 5 amino acids to about 95 amino acids,about 5 amino acids to about 90 amino acids, about 5 amino acids toabout 85 amino acids, about 5 amino acids to about 80 amino acids, about5 amino acids to about 75 amino acids, about 5 amino acids to about 70amino acids, about 5 amino acids to about 65 amino acids, about 5 aminoacids to about 60 amino acids, about 5 amino acids to about 55 aminoacids, about 5 amino acids to about 50 amino acids, about 5 amino acidsto about 45 amino acids, about 5 amino acids to about 40 amino acids,about 5 amino acids to about 35 amino acids, about 5 amino acids toabout 30 amino acids, about 5 amino acids to about 25 amino acids, about5 amino acids to about 20 amino acids, about 5 amino acids to about 15amino acids, about 5 amino acids to about 10 amino acids, about 10 aminoacids to about 1000 amino acids, about 10 amino acids to about 950 aminoacids, about 10 amino acids to about 900 amino acids, about 10 aminoacids to about 850 amino acids, about 10 amino acids to about 800 aminoacids, about 10 amino acids to about 750 amino acids, about 10 aminoacids to about 700 amino acids, about 10 amino acids to about 650 aminoacids, about 10 amino acids to about 600 amino acids, about 10 aminoacids to about 550 amino acids, about 10 amino acids to about 500 aminoacids, about 10 amino acids to about 450 amino acids, about 10 aminoacids to about 400 amino acids, about 10 amino acids to about 350 aminoacids, about 10 amino acids to about 300 amino acids, about 10 aminoacids to about 280 amino acids, about 10 amino acids to about 260 aminoacids, about 10 amino acids to about 240 amino acids, about 10 aminoacids to about 220 amino acids, about 10 amino acids to about 200 aminoacids, about 10 amino acids to about 195 amino acids, about 10 aminoacids to about 190 amino acids, about 10 amino acids to about 185 aminoacids, about 10 amino acids to about 180 amino acids, about 10 aminoacids to about 175 amino acids, about 10 amino acids to about 170 aminoacids, about 10 amino acids to about 165 amino acids, about 10 aminoacids to about 160 amino acids, about 10 amino acids to about 155 aminoacids, about 10 amino acids to about 150 amino acids, about 10 aminoacids to about 145 amino acids, about 10 amino acids to about 140 aminoacids, about 10 amino acids to about 135 amino acids, about 10 aminoacids to about 130 amino acids, about 10 amino acids to about 125 aminoacids, about 10 amino acids to about 120 amino acids, about 10 aminoacids to about 115 amino acids, about 10 amino acids to about 110 aminoacids, about 10 amino acids to about 105 amino acids, about 10 aminoacids to about 100 amino acids, about 10 amino acids to about 95 aminoacids, about 10 amino acids to about 90 amino acids, about 10 aminoacids to about 85 amino acids, about 10 amino acids to about 80 aminoacids, about 10 amino acids to about 75 amino acids, about 10 aminoacids to about 70 amino acids, about 10 amino acids to about 65 aminoacids, about 10 amino acids to about 60 amino acids, about 10 aminoacids to about 55 amino acids, about 10 amino acids to about 50 aminoacids, about 10 amino acids to about 45 amino acids, about 10 aminoacids to about 40 amino acids, about 10 amino acids to about 35 aminoacids, about 10 amino acids to about 30 amino acids, about 10 aminoacids to about 25 amino acids, about 10 amino acids to about 20 aminoacids, about 10 amino acids to about 15 amino acids, about 15 aminoacids to about 1000 amino acids, about 15 amino acids to about 950 aminoacids, about 15 amino acids to about 900 amino acids, about 15 aminoacids to about 850 amino acids, about 15 amino acids to about 800 aminoacids, about 15 amino acids to about 750 amino acids, about 15 aminoacids to about 700 amino acids, about 15 amino acids to about 650 aminoacids, about 15 amino acids to about 600 amino acids, about 15 aminoacids to about 550 amino acids, about 15 amino acids to about 500 aminoacids, about 15 amino acids to about 450 amino acids, about 15 aminoacids to about 400 amino acids, about 15 amino acids to about 350 aminoacids, about 15 amino acids to about 300 amino acids, about 15 aminoacids to about 280 amino acids, about 15 amino acids to about 260 aminoacids, about 15 amino acids to about 240 amino acids, about 15 aminoacids to about 220 amino acids, about 15 amino acids to about 200 aminoacids, about 15 amino acids to about 195 amino acids, about 15 aminoacids to about 190 amino acids, about 15 amino acids to about 185 aminoacids, about 15 amino acids to about 180 amino acids, about 15 aminoacids to about 175 amino acids, about 15 amino acids to about 170 aminoacids, about 15 amino acids to about 165 amino acids, about 15 aminoacids to about 160 amino acids, about 15 amino acids to about 155 aminoacids, about 15 amino acids to about 150 amino acids, about 15 aminoacids to about 145 amino acids, about 15 amino acids to about 140 aminoacids, about 15 amino acids to about 135 amino acids, about 15 aminoacids to about 130 amino acids, about 15 amino acids to about 125 aminoacids, about 15 amino acids to about 120 amino acids, about 15 aminoacids to about 115 amino acids, about 15 amino acids to about 110 aminoacids, about 15 amino acids to about 105 amino acids, about 15 aminoacids to about 100 amino acids, about 15 amino acids to about 95 aminoacids, about 15 amino acids to about 90 amino acids, about 15 aminoacids to about 85 amino acids, about 15 amino acids to about 80 aminoacids, about 15 amino acids to about 75 amino acids, about 15 aminoacids to about 70 amino acids, about 15 amino acids to about 65 aminoacids, about 15 amino acids to about 60 amino acids, about 15 aminoacids to about 55 amino acids, about 15 amino acids to about 50 aminoacids, about 15 amino acids to about 45 amino acids, about 15 aminoacids to about 40 amino acids, about 15 amino acids to about 35 aminoacids, about 15 amino acids to about 30 amino acids, about 15 aminoacids to about 25 amino acids, about 15 amino acids to about 20 aminoacids, about 20 amino acids to about 1000 amino acids, about 20 aminoacids to about 950 amino acids, about 20 amino acids to about 900 aminoacids, about 20 amino acids to about 850 amino acids, about 20 aminoacids to about 800 amino acids, about 20 amino acids to about 750 aminoacids, about 20 amino acids to about 700 amino acids, about 20 aminoacids to about 650 amino acids, about 20 amino acids to about 600 aminoacids, about 20 amino acids to about 550 amino acids, about 20 aminoacids to about 500 amino acids, about 20 amino acids to about 450 aminoacids, about 20 amino acids to about 400 amino acids, about 20 aminoacids to about 350 amino acids, about 20 amino acids to about 300 aminoacids, about 20 amino acids to about 280 amino acids, about 20 aminoacids to about 260 amino acids, about 20 amino acids to about 240 aminoacids, about 20 amino acids to about 220 amino acids, about 20 aminoacids to about 200 amino acids, about 20 amino acids to about 195 aminoacids, about 20 amino acids to about 190 amino acids, about 20 aminoacids to about 185 amino acids, about 20 amino acids to about 180 aminoacids, about 20 amino acids to about 175 amino acids, about 20 aminoacids to about 170 amino acids, about 20 amino acids to about 165 aminoacids, about 20 amino acids to about 160 amino acids, about 20 aminoacids to about 155 amino acids, about 20 amino acids to about 150 aminoacids, about 20 amino acids to about 145 amino acids, about 20 aminoacids to about 140 amino acids, about 20 amino acids to about 135 aminoacids, about 20 amino acids to about 130 amino acids, about 20 aminoacids to about 125 amino acids, about 20 amino acids to about 120 aminoacids, about 20 amino acids to about 115 amino acids, about 20 aminoacids to about 110 amino acids, about 20 amino acids to about 105 aminoacids, about 20 amino acids to about 100 amino acids, about 20 aminoacids to about 95 amino acids, about 20 amino acids to about 90 aminoacids, about 20 amino acids to about 85 amino acids, about 20 aminoacids to about 80 amino acids, about 20 amino acids to about 75 aminoacids, about 20 amino acids to about 70 amino acids, about 20 aminoacids to about 65 amino acids, about 20 amino acids to about 60 aminoacids, about 20 amino acids to about 55 amino acids, about 20 aminoacids to about 50 amino acids, about 20 amino acids to about 45 aminoacids, about 20 amino acids to about 40 amino acids, about 20 aminoacids to about 35 amino acids, about 20 amino acids to about 30 aminoacids, about 20 amino acids to about 25 amino acids, about 25 aminoacids to about 1000 amino acids, about 25 amino acids to about 950 aminoacids, about 25 amino acids to about 900 amino acids, about 25 aminoacids to about 850 amino acids, about 25 amino acids to about 800 aminoacids, about 25 amino acids to about 750 amino acids, about 25 aminoacids to about 700 amino acids, about 25 amino acids to about 650 aminoacids, about 25 amino acids to about 600 amino acids, about 25 aminoacids to about 550 amino acids, about 25 amino acids to about 500 aminoacids, about 25 amino acids to about 450 amino acids, about 25 aminoacids to about 400 amino acids, about 25 amino acids to about 350 aminoacids, about 25 amino acids to about 300 amino acids, about 25 aminoacids to about 280 amino acids, about 25 amino acids to about 260 aminoacids, about 25 amino acids to about 240 amino acids, about 25 aminoacids to about 220 amino acids, about 25 amino acids to about 200 aminoacids, about 25 amino acids to about 195 amino acids, about 25 aminoacids to about 190 amino acids, about 25 amino acids to about 185 aminoacids, about 25 amino acids to about 180 amino acids, about 25 aminoacids to about 175 amino acids, about 25 amino acids to about 170 aminoacids, about 25 amino acids to about 165 amino acids, about 25 aminoacids to about 160 amino acids, about 25 amino acids to about 155 aminoacids, about 25 amino acids to about 150 amino acids, about 25 aminoacids to about 145 amino acids, about 25 amino acids to about 140 aminoacids, about 25 amino acids to about 135 amino acids, about 25 aminoacids to about 130 amino acids, about 25 amino acids to about 125 aminoacids, about 25 amino acids to about 120 amino acids, about 25 aminoacids to about 115 amino acids, about 25 amino acids to about 110 aminoacids, about 25 amino acids to about 105 amino acids, about 25 aminoacids to about 100 amino acids, about 25 amino acids to about 95 aminoacids, about 25 amino acids to about 90 amino acids, about 25 aminoacids to about 85 amino acids, about 25 amino acids to about 80 aminoacids, about 25 amino acids to about 75 amino acids, about 25 aminoacids to about 70 amino acids, about 25 amino acids to about 65 aminoacids, about 25 amino acids to about 60 amino acids, about 25 aminoacids to about 55 amino acids, about 25 amino acids to about 50 aminoacids, about 25 amino acids to about 45 amino acids, about 25 aminoacids to about 40 amino acids, about 25 amino acids to about 35 aminoacids, about 25 amino acids to about 30 amino acids, about 30 aminoacids to about 1000 amino acids, about 30 amino acids to about 950 aminoacids, about 30 amino acids to about 900 amino acids, about 30 aminoacids to about 850 amino acids, about 30 amino acids to about 800 aminoacids, about 30 amino acids to about 750 amino acids, about 30 aminoacids to about 700 amino acids, about 30 amino acids to about 650 aminoacids, about 30 amino acids to about 600 amino acids, about 30 aminoacids to about 550 amino acids, about 30 amino acids to about 500 aminoacids, about 30 amino acids to about 450 amino acids, about 30 aminoacids to about 400 amino acids, about 30 amino acids to about 350 aminoacids, about 30 amino acids to about 300 amino acids, about 30 aminoacids to about 280 amino acids, about 30 amino acids to about 260 aminoacids, about 30 amino acids to about 240 amino acids, about 30 aminoacids to about 220 amino acids, about 30 amino acids to about 200 aminoacids, about 30 amino acids to about 195 amino acids, about 30 aminoacids to about 190 amino acids, about 30 amino acids to about 185 aminoacids, about 30 amino acids to about 180 amino acids, about 30 aminoacids to about 175 amino acids, about 30 amino acids to about 170 aminoacids, about 30 amino acids to about 165 amino acids, about 30 aminoacids to about 160 amino acids, about 30 amino acids to about 155 aminoacids, about 30 amino acids to about 150 amino acids, about 30 aminoacids to about 145 amino acids, about 30 amino acids to about 140 aminoacids, about 30 amino acids to about 135 amino acids, about 30 aminoacids to about 130 amino acids, about 30 amino acids to about 125 aminoacids, about 30 amino acids to about 120 amino acids, about 30 aminoacids to about 115 amino acids, about 30 amino acids to about 110 aminoacids, about 30 amino acids to about 105 amino acids, about 30 aminoacids to about 100 amino acids, about 30 amino acids to about 95 aminoacids, about 30 amino acids to about 90 amino acids, about 30 aminoacids to about 85 amino acids, about 30 amino acids to about 80 aminoacids, about 30 amino acids to about 75 amino acids, about 30 aminoacids to about 70 amino acids, about 30 amino acids to about 65 aminoacids, about 30 amino acids to about 60 amino acids, about 30 aminoacids to about 55 amino acids, about 30 amino acids to about 50 aminoacids, about 30 amino acids to about 45 amino acids, about 30 aminoacids to about 40 amino acids, about 30 amino acids to about 35 aminoacids, about 35 amino acids to about 1000 amino acids, about 35 aminoacids to about 950 amino acids, about 35 amino acids to about 900 aminoacids, about 35 amino acids to about 850 amino acids, about 35 aminoacids to about 800 amino acids, about 35 amino acids to about 750 aminoacids, about 35 amino acids to about 700 amino acids, about 35 aminoacids to about 650 amino acids, about 35 amino acids to about 600 aminoacids, about 35 amino acids to about 550 amino acids, about 35 aminoacids to about 500 amino acids, about 35 amino acids to about 450 aminoacids, about 35 amino acids to about 400 amino acids, about 35 aminoacids to about 350 amino acids, about 35 amino acids to about 300 aminoacids, about 35 amino acids to about 280 amino acids, about 35 aminoacids to about 260 amino acids, about 35 amino acids to about 240 aminoacids, about 35 amino acids to about 220 amino acids, about 35 aminoacids to about 200 amino acids, about 35 amino acids to about 195 aminoacids, about 35 amino acids to about 190 amino acids, about 35 aminoacids to about 185 amino acids, about 35 amino acids to about 180 aminoacids, about 35 amino acids to about 175 amino acids, about 35 aminoacids to about 170 amino acids, about 35 amino acids to about 165 aminoacids, about 35 amino acids to about 160 amino acids, about 35 aminoacids to about 155 amino acids, about 35 amino acids to about 150 aminoacids, about 35 amino acids to about 145 amino acids, about 35 aminoacids to about 140 amino acids, about 35 amino acids to about 135 aminoacids, about 35 amino acids to about 130 amino acids, about 35 aminoacids to about 125 amino acids, about 35 amino acids to about 120 aminoacids, about 35 amino acids to about 115 amino acids, about 35 aminoacids to about 110 amino acids, about 35 amino acids to about 105 aminoacids, about 35 amino acids to about 100 amino acids, about 35 aminoacids to about 95 amino acids, about 35 amino acids to about 90 aminoacids, about 35 amino acids to about 85 amino acids, about 35 aminoacids to about 80 amino acids, about 35 amino acids to about 75 aminoacids, about 35 amino acids to about 70 amino acids, about 35 aminoacids to about 65 amino acids, about 35 amino acids to about 60 aminoacids, about 35 amino acids to about 55 amino acids, about 35 aminoacids to about 50 amino acids, about 35 amino acids to about 45 aminoacids, about 35 amino acids to about 40 amino acids, about 40 aminoacids to about 1000 amino acids, about 40 amino acids to about 950 aminoacids, about 40 amino acids to about 900 amino acids, about 40 aminoacids to about 850 amino acids, about 40 amino acids to about 800 aminoacids, about 40 amino acids to about 750 amino acids, about 40 aminoacids to about 700 amino acids, about 40 amino acids to about 650 aminoacids, about 40 amino acids to about 600 amino acids, about 40 aminoacids to about 550 amino acids, about 40 amino acids to about 500 aminoacids, about 40 amino acids to about 450 amino acids, about 40 aminoacids to about 400 amino acids, about 40 amino acids to about 350 aminoacids, about 40 amino acids to about 300 amino acids, about 40 aminoacids to about 280 amino acids, about 40 amino acids to about 260 aminoacids, about 40 amino acids to about 240 amino acids, about 40 aminoacids to about 220 amino acids, about 40 amino acids to about 200 aminoacids, about 40 amino acids to about 195 amino acids, about 40 aminoacids to about 190 amino acids, about 40 amino acids to about 185 aminoacids, about 40 amino acids to about 180 amino acids, about 40 aminoacids to about 175 amino acids, about 40 amino acids to about 170 aminoacids, about 40 amino acids to about 165 amino acids, about 40 aminoacids to about 160 amino acids, about 40 amino acids to about 155 aminoacids, about 40 amino acids to about 150 amino acids, about 40 aminoacids to about 145 amino acids, about 40 amino acids to about 140 aminoacids, about 40 amino acids to about 135 amino acids, about 40 aminoacids to about 130 amino acids, about 40 amino acids to about 125 aminoacids, about 40 amino acids to about 120 amino acids, about 40 aminoacids to about 115 amino acids, about 40 amino acids to about 110 aminoacids, about 40 amino acids to about 105 amino acids, about 40 aminoacids to about 100 amino acids, about 40 amino acids to about 95 aminoacids, about 40 amino acids to about 90 amino acids, about 40 aminoacids to about 85 amino acids, about 40 amino acids to about 80 aminoacids, about 40 amino acids to about 75 amino acids, about 40 aminoacids to about 70 amino acids, about 40 amino acids to about 65 aminoacids, about 40 amino acids to about 60 amino acids, about 40 aminoacids to about 55 amino acids, about 40 amino acids to about 50 aminoacids, about 40 amino acids to about 45 amino acids, about 45 aminoacids to about 1000 amino acids, about 45 amino acids to about 950 aminoacids, about 45 amino acids to about 900 amino acids, about 45 aminoacids to about 850 amino acids, about 45 amino acids to about 800 aminoacids, about 45 amino acids to about 750 amino acids, about 45 aminoacids to about 700 amino acids, about 45 amino acids to about 650 aminoacids, about 45 amino acids to about 600 amino acids, about 45 aminoacids to about 550 amino acids, about 45 amino acids to about 500 aminoacids, about 45 amino acids to about 450 amino acids, about 45 aminoacids to about 400 amino acids, about 45 amino acids to about 350 aminoacids, about 45 amino acids to about 300 amino acids, about 45 aminoacids to about 280 amino acids, about 45 amino acids to about 260 aminoacids, about 45 amino acids to about 240 amino acids, about 45 aminoacids to about 220 amino acids, about 45 amino acids to about 200 aminoacids, about 45 amino acids to about 195 amino acids, about 45 aminoacids to about 190 amino acids, about 45 amino acids to about 185 aminoacids, about 45 amino acids to about 180 amino acids, about 45 aminoacids to about 175 amino acids, about 45 amino acids to about 170 aminoacids, about 45 amino acids to about 165 amino acids, about 45 aminoacids to about 160 amino acids, about 45 amino acids to about 155 aminoacids, about 45 amino acids to about 150 amino acids, about 45 aminoacids to about 145 amino acids, about 45 amino acids to about 140 aminoacids, about 45 amino acids to about 135 amino acids, about 45 aminoacids to about 130 amino acids, about 45 amino acids to about 125 aminoacids, about 45 amino acids to about 120 amino acids, about 45 aminoacids to about 115 amino acids, about 45 amino acids to about 110 aminoacids, about 45 amino acids to about 105 amino acids, about 45 aminoacids to about 100 amino acids, about 45 amino acids to about 95 aminoacids, about 45 amino acids to about 90 amino acids, about 45 aminoacids to about 85 amino acids, about 45 amino acids to about 80 aminoacids, about 45 amino acids to about 75 amino acids, about 45 aminoacids to about 70 amino acids, about 45 amino acids to about 65 aminoacids, about 45 amino acids to about 60 amino acids, about 45 aminoacids to about 55 amino acids, about 45 amino acids to about 50 aminoacids, about 50 amino acids to about 1000 amino acids, about 50 aminoacids to about 950 amino acids, about 50 amino acids to about 900 aminoacids, about 50 amino acids to about 850 amino acids, about 50 aminoacids to about 800 amino acids, about 50 amino acids to about 750 aminoacids, about 50 amino acids to about 700 amino acids, about 50 aminoacids to about 650 amino acids, about 50 amino acids to about 600 aminoacids, about 50 amino acids to about 550 amino acids, about 50 aminoacids to about 500 amino acids, about 50 amino acids to about 450 aminoacids, about 50 amino acids to about 400 amino acids, about 50 aminoacids to about 350 amino acids, about 50 amino acids to about 300 aminoacids, about 50 amino acids to about 280 amino acids, about 50 aminoacids to about 260 amino acids, about 50 amino acids to about 240 aminoacids, about 50 amino acids to about 220 amino acids, about 50 aminoacids to about 200 amino acids, about 50 amino acids to about 195 aminoacids, about 50 amino acids to about 190 amino acids, about 50 aminoacids to about 185 amino acids, about 50 amino acids to about 180 aminoacids, about 50 amino acids to about 175 amino acids, about 50 aminoacids to about 170 amino acids, about 50 amino acids to about 165 aminoacids, about 50 amino acids to about 160 amino acids, about 50 aminoacids to about 155 amino acids, about 50 amino acids to about 150 aminoacids, about 50 amino acids to about 145 amino acids, about 50 aminoacids to about 140 amino acids, about 50 amino acids to about 135 aminoacids, about 50 amino acids to about 130 amino acids, about 50 aminoacids to about 125 amino acids, about 50 amino acids to about 120 aminoacids, about 50 amino acids to about 115 amino acids, about 50 aminoacids to about 110 amino acids, about 50 amino acids to about 105 aminoacids, about 50 amino acids to about 100 amino acids, about 50 aminoacids to about 95 amino acids, about 50 amino acids to about 90 aminoacids, about 50 amino acids to about 85 amino acids, about 50 aminoacids to about 80 amino acids, about 50 amino acids to about 75 aminoacids, about 50 amino acids to about 70 amino acids, about 50 aminoacids to about 65 amino acids, about 50 amino acids to about 60 aminoacids, about 50 amino acids to about 55 amino acids, about 55 aminoacids to about 1000 amino acids, about 55 amino acids to about 950 aminoacids, about 55 amino acids to about 900 amino acids, about 55 aminoacids to about 850 amino acids, about 55 amino acids to about 800 aminoacids, about 55 amino acids to about 750 amino acids, about 55 aminoacids to about 700 amino acids, about 55 amino acids to about 650 aminoacids, about 55 amino acids to about 600 amino acids, about 55 aminoacids to about 550 amino acids, about 55 amino acids to about 500 aminoacids, about 55 amino acids to about 450 amino acids, about 55 aminoacids to about 400 amino acids, about 55 amino acids to about 350 aminoacids, about 55 amino acids to about 300 amino acids, about 55 aminoacids to about 280 amino acids, about 55 amino acids to about 260 aminoacids, about 55 amino acids to about 240 amino acids, about 55 aminoacids to about 220 amino acids, about 55 amino acids to about 200 aminoacids, about 55 amino acids to about 195 amino acids, about 55 aminoacids to about 190 amino acids, about 55 amino acids to about 185 aminoacids, about 55 amino acids to about 180 amino acids, about 55 aminoacids to about 175 amino acids, about 55 amino acids to about 170 aminoacids, about 55 amino acids to about 165 amino acids, about 55 aminoacids to about 160 amino acids, about 55 amino acids to about 155 aminoacids, about 55 amino acids to about 150 amino acids, about 55 aminoacids to about 145 amino acids, about 55 amino acids to about 140 aminoacids, about 55 amino acids to about 135 amino acids, about 55 aminoacids to about 130 amino acids, about 55 amino acids to about 125 aminoacids, about 55 amino acids to about 120 amino acids, about 55 aminoacids to about 115 amino acids, about 55 amino acids to about 110 aminoacids, about 55 amino acids to about 105 amino acids, about 55 aminoacids to about 100 amino acids, about 55 amino acids to about 95 aminoacids, about 55 amino acids to about 90 amino acids, about 55 aminoacids to about 85 amino acids, about 55 amino acids to about 80 aminoacids, about 55 amino acids to about 75 amino acids, about 55 aminoacids to about 70 amino acids, about 55 amino acids to about 65 aminoacids, about 55 amino acids to about 60 amino acids, about 60 aminoacids to about 1000 amino acids, about 60 amino acids to about 950 aminoacids, about 60 amino acids to about 900 amino acids, about 60 aminoacids to about 850 amino acids, about 60 amino acids to about 800 aminoacids, about 60 amino acids to about 750 amino acids, about 60 aminoacids to about 700 amino acids, about 60 amino acids to about 650 aminoacids, about 60 amino acids to about 600 amino acids, about 60 aminoacids to about 550 amino acids, about 60 amino acids to about 500 aminoacids, about 60 amino acids to about 450 amino acids, about 60 aminoacids to about 400 amino acids, about 60 amino acids to about 350 aminoacids, about 60 amino acids to about 300 amino acids, about 60 aminoacids to about 280 amino acids, about 60 amino acids to about 260 aminoacids, about 60 amino acids to about 240 amino acids, about 60 aminoacids to about 220 amino acids, about 60 amino acids to about 200 aminoacids, about 60 amino acids to about 195 amino acids, about 60 aminoacids to about 190 amino acids, about 60 amino acids to about 185 aminoacids, about 60 amino acids to about 180 amino acids, about 60 aminoacids to about 175 amino acids, about 60 amino acids to about 170 aminoacids, about 60 amino acids to about 165 amino acids, about 60 aminoacids to about 160 amino acids, about 60 amino acids to about 155 aminoacids, about 60 amino acids to about 150 amino acids, about 60 aminoacids to about 145 amino acids, about 60 amino acids to about 140 aminoacids, about 60 amino acids to about 135 amino acids, about 60 aminoacids to about 130 amino acids, about 60 amino acids to about 125 aminoacids, about 60 amino acids to about 120 amino acids, about 60 aminoacids to about 115 amino acids, about 60 amino acids to about 110 aminoacids, about 60 amino acids to about 105 amino acids, about 60 aminoacids to about 100 amino acids, about 60 amino acids to about 95 aminoacids, about 60 amino acids to about 90 amino acids, about 60 aminoacids to about 85 amino acids, about 60 amino acids to about 80 aminoacids, about 60 amino acids to about 75 amino acids, about 60 aminoacids to about 70 amino acids, about 60 amino acids to about 65 aminoacids, about 65 amino acids to about 1000 amino acids, about 65 aminoacids to about 950 amino acids, about 65 amino acids to about 900 aminoacids, about 65 amino acids to about 850 amino acids, about 65 aminoacids to about 800 amino acids, about 65 amino acids to about 750 aminoacids, about 65 amino acids to about 700 amino acids, about 65 aminoacids to about 650 amino acids, about 65 amino acids to about 600 aminoacids, about 65 amino acids to about 550 amino acids, about 65 aminoacids to about 500 amino acids, about 65 amino acids to about 450 aminoacids, about 65 amino acids to about 400 amino acids, about 65 aminoacids to about 350 amino acids, about 65 amino acids to about 300 aminoacids, about 65 amino acids to about 280 amino acids, about 65 aminoacids to about 260 amino acids, about 65 amino acids to about 240 aminoacids, about 65 amino acids to about 220 amino acids, about 65 aminoacids to about 200 amino acids, about 65 amino acids to about 195 aminoacids, about 65 amino acids to about 190 amino acids, about 65 aminoacids to about 185 amino acids, about 65 amino acids to about 180 aminoacids, about 65 amino acids to about 175 amino acids, about 65 aminoacids to about 170 amino acids, about 65 amino acids to about 165 aminoacids, about 65 amino acids to about 160 amino acids, about 65 aminoacids to about 155 amino acids, about 65 amino acids to about 150 aminoacids, about 65 amino acids to about 145 amino acids, about 65 aminoacids to about 140 amino acids, about 65 amino acids to about 135 aminoacids, about 65 amino acids to about 130 amino acids, about 65 aminoacids to about 125 amino acids, about 65 amino acids to about 120 aminoacids, about 65 amino acids to about 115 amino acids, about 65 aminoacids to about 110 amino acids, about 65 amino acids to about 105 aminoacids, about 65 amino acids to about 100 amino acids, about 65 aminoacids to about 95 amino acids, about 65 amino acids to about 90 aminoacids, about 65 amino acids to about 85 amino acids, about 65 aminoacids to about 80 amino acids, about 65 amino acids to about 75 aminoacids, about 65 amino acids to about 70 amino acids, about 70 aminoacids to about 1000 amino acids, about 70 amino acids to about 950 aminoacids, about 70 amino acids to about 900 amino acids, about 70 aminoacids to about 850 amino acids, about 70 amino acids to about 800 aminoacids, about 70 amino acids to about 750 amino acids, about 70 aminoacids to about 700 amino acids, about 70 amino acids to about 650 aminoacids, about 70 amino acids to about 600 amino acids, about 70 aminoacids to about 550 amino acids, about 70 amino acids to about 500 aminoacids, about 70 amino acids to about 450 amino acids, about 70 aminoacids to about 400 amino acids, about 70 amino acids to about 350 aminoacids, about 70 amino acids to about 300 amino acids, about 70 aminoacids to about 280 amino acids, about 70 amino acids to about 260 aminoacids, about 70 amino acids to about 240 amino acids, about 70 aminoacids to about 220 amino acids, about 70 amino acids to about 200 aminoacids, about 70 amino acids to about 195 amino acids, about 70 aminoacids to about 190 amino acids, about 70 amino acids to about 185 aminoacids, about 70 amino acids to about 180 amino acids, about 70 aminoacids to about 175 amino acids, about 70 amino acids to about 170 aminoacids, about 70 amino acids to about 165 amino acids, about 70 aminoacids to about 160 amino acids, about 70 amino acids to about 155 aminoacids, about 70 amino acids to about 150 amino acids, about 70 aminoacids to about 145 amino acids, about 70 amino acids to about 140 aminoacids, about 70 amino acids to about 135 amino acids, about 70 aminoacids to about 130 amino acids, about 70 amino acids to about 125 aminoacids, about 70 amino acids to about 120 amino acids, about 70 aminoacids to about 115 amino acids, about 70 amino acids to about 110 aminoacids, about 70 amino acids to about 105 amino acids, about 70 aminoacids to about 100 amino acids, about 70 amino acids to about 95 aminoacids, about 70 amino acids to about 90 amino acids, about 70 aminoacids to about 85 amino acids, about 70 amino acids to about 80 aminoacids, about 70 amino acids to about 75 amino acids, about 75 aminoacids to about 1000 amino acids, about 75 amino acids to about 950 aminoacids, about 75 amino acids to about 900 amino acids, about 75 aminoacids to about 850 amino acids, about 75 amino acids to about 800 aminoacids, about 75 amino acids to about 750 amino acids, about 75 aminoacids to about 700 amino acids, about 75 amino acids to about 650 aminoacids, about 75 amino acids to about 600 amino acids, about 75 aminoacids to about 550 amino acids, about 75 amino acids to about 500 aminoacids, about 75 amino acids to about 450 amino acids, about 75 aminoacids to about 400 amino acids, about 75 amino acids to about 350 aminoacids, about 75 amino acids to about 300 amino acids, about 75 aminoacids to about 280 amino acids, about 75 amino acids to about 260 aminoacids, about 75 amino acids to about 240 amino acids, about 75 aminoacids to about 220 amino acids, about 75 amino acids to about 200 aminoacids, about 75 amino acids to about 195 amino acids, about 75 aminoacids to about 190 amino acids, about 75 amino acids to about 185 aminoacids, about 75 amino acids to about 180 amino acids, about 75 aminoacids to about 175 amino acids, about 75 amino acids to about 170 aminoacids, about 75 amino acids to about 165 amino acids, about 75 aminoacids to about 160 amino acids, about 75 amino acids to about 155 aminoacids, about 75 amino acids to about 150 amino acids, about 75 aminoacids to about 145 amino acids, about 75 amino acids to about 140 aminoacids, about 75 amino acids to about 135 amino acids, about 75 aminoacids to about 130 amino acids, about 75 amino acids to about 125 aminoacids, about 75 amino acids to about 120 amino acids, about 75 aminoacids to about 115 amino acids, about 75 amino acids to about 110 aminoacids, about 75 amino acids to about 105 amino acids, about 75 aminoacids to about 100 amino acids, about 75 amino acids to about 95 aminoacids, about 75 amino acids to about 90 amino acids, about 75 aminoacids to about 85 amino acids, about 75 amino acids to about 80 aminoacids, about 80 amino acids to about 1000 amino acids, about 80 aminoacids to about 950 amino acids, about 80 amino acids to about 900 aminoacids, about 80 amino acids to about 850 amino acids, about 80 aminoacids to about 800 amino acids, about 80 amino acids to about 750 aminoacids, about 80 amino acids to about 700 amino acids, about 80 aminoacids to about 650 amino acids, about 80 amino acids to about 600 aminoacids, about 80 amino acids to about 550 amino acids, about 80 aminoacids to about 500 amino acids, about 80 amino acids to about 450 aminoacids, about 80 amino acids to about 400 amino acids, about 80 aminoacids to about 350 amino acids, about 80 amino acids to about 300 aminoacids, about 80 amino acids to about 280 amino acids, about 80 aminoacids to about 260 amino acids, about 80 amino acids to about 240 aminoacids, about 80 amino acids to about 220 amino acids, about 80 aminoacids to about 200 amino acids, about 80 amino acids to about 195 aminoacids, about 80 amino acids to about 190 amino acids, about 80 aminoacids to about 185 amino acids, about 80 amino acids to about 180 aminoacids, about 80 amino acids to about 175 amino acids, about 80 aminoacids to about 170 amino acids, about 80 amino acids to about 165 aminoacids, about 80 amino acids to about 160 amino acids, about 80 aminoacids to about 155 amino acids, about 80 amino acids to about 150 aminoacids, about 80 amino acids to about 145 amino acids, about 80 aminoacids to about 140 amino acids, about 80 amino acids to about 135 aminoacids, about 80 amino acids to about 130 amino acids, about 80 aminoacids to about 125 amino acids, about 80 amino acids to about 120 aminoacids, about 80 amino acids to about 115 amino acids, about 80 aminoacids to about 110 amino acids, about 80 amino acids to about 105 aminoacids, about 80 amino acids to about 100 amino acids, about 80 aminoacids to about 95 amino acids, about 80 amino acids to about 90 aminoacids, about 80 amino acids to about 85 amino acids, about 85 aminoacids to about 1000 amino acids, about 85 amino acids to about 950 aminoacids, about 85 amino acids to about 900 amino acids, about 85 aminoacids to about 850 amino acids, about 85 amino acids to about 800 aminoacids, about 85 amino acids to about 750 amino acids, about 85 aminoacids to about 700 amino acids, about 85 amino acids to about 650 aminoacids, about 85 amino acids to about 600 amino acids, about 85 aminoacids to about 550 amino acids, about 85 amino acids to about 500 aminoacids, about 85 amino acids to about 450 amino acids, about 85 aminoacids to about 400 amino acids, about 85 amino acids to about 350 aminoacids, about 85 amino acids to about 300 amino acids, about 85 aminoacids to about 280 amino acids, about 85 amino acids to about 260 aminoacids, about 85 amino acids to about 240 amino acids, about 85 aminoacids to about 220 amino acids, about 85 amino acids to about 200 aminoacids, about 85 amino acids to about 195 amino acids, about 85 aminoacids to about 190 amino acids, about 85 amino acids to about 185 aminoacids, about 85 amino acids to about 180 amino acids, about 85 aminoacids to about 175 amino acids, about 85 amino acids to about 170 aminoacids, about 85 amino acids to about 165 amino acids, about 85 aminoacids to about 160 amino acids, about 85 amino acids to about 155 aminoacids, about 85 amino acids to about 150 amino acids, about 85 aminoacids to about 145 amino acids, about 85 amino acids to about 140 aminoacids, about 85 amino acids to about 135 amino acids, about 85 aminoacids to about 130 amino acids, about 85 amino acids to about 125 aminoacids, about 85 amino acids to about 120 amino acids, about 85 aminoacids to about 115 amino acids, about 85 amino acids to about 110 aminoacids, about 85 amino acids to about 105 amino acids, about 85 aminoacids to about 100 amino acids, about 85 amino acids to about 95 aminoacids, about 85 amino acids to about 90 amino acids, about 90 aminoacids to about 1000 amino acids, about 90 amino acids to about 950 aminoacids, about 90 amino acids to about 900 amino acids, about 90 aminoacids to about 850 amino acids, about 90 amino acids to about 800 aminoacids, about 90 amino acids to about 750 amino acids, about 90 aminoacids to about 700 amino acids, about 90 amino acids to about 650 aminoacids, about 90 amino acids to about 600 amino acids, about 90 aminoacids to about 550 amino acids, about 90 amino acids to about 500 aminoacids, about 90 amino acids to about 450 amino acids, about 90 aminoacids to about 400 amino acids, about 90 amino acids to about 350 aminoacids, about 90 amino acids to about 300 amino acids, about 90 aminoacids to about 280 amino acids, about 90 amino acids to about 260 aminoacids, about 90 amino acids to about 240 amino acids, about 90 aminoacids to about 220 amino acids, about 90 amino acids to about 200 aminoacids, about 90 amino acids to about 195 amino acids, about 90 aminoacids to about 190 amino acids, about 90 amino acids to about 185 aminoacids, about 90 amino acids to about 180 amino acids, about 90 aminoacids to about 175 amino acids, about 90 amino acids to about 170 aminoacids, about 90 amino acids to about 165 amino acids, about 90 aminoacids to about 160 amino acids, about 90 amino acids to about 155 aminoacids, about 90 amino acids to about 150 amino acids, about 90 aminoacids to about 145 amino acids, about 90 amino acids to about 140 aminoacids, about 90 amino acids to about 135 amino acids, about 90 aminoacids to about 130 amino acids, about 90 amino acids to about 125 aminoacids, about 90 amino acids to about 120 amino acids, about 90 aminoacids to about 115 amino acids, about 90 amino acids to about 110 aminoacids, about 90 amino acids to about 105 amino acids, about 90 aminoacids to about 100 amino acids, about 90 amino acids to about 95 aminoacids, about 95 amino acids to about 1000 amino acids, about 95 aminoacids to about 950 amino acids, about 95 amino acids to about 900 aminoacids, about 95 amino acids to about 850 amino acids, about 95 aminoacids to about 800 amino acids, about 95 amino acids to about 750 aminoacids, about 95 amino acids to about 700 amino acids, about 95 aminoacids to about 650 amino acids, about 95 amino acids to about 600 aminoacids, about 95 amino acids to about 550 amino acids, about 95 aminoacids to about 500 amino acids, about 95 amino acids to about 450 aminoacids, about 95 amino acids to about 400 amino acids, about 95 aminoacids to about 350 amino acids, about 95 amino acids to about 300 aminoacids, about 95 amino acids to about 280 amino acids, about 95 aminoacids to about 260 amino acids, about 95 amino acids to about 240 aminoacids, about 95 amino acids to about 220 amino acids, about 95 aminoacids to about 200 amino acids, about 95 amino acids to about 195 aminoacids, about 95 amino acids to about 190 amino acids, about 95 aminoacids to about 185 amino acids, about 95 amino acids to about 180 aminoacids, about 95 amino acids to about 175 amino acids, about 95 aminoacids to about 170 amino acids, about 95 amino acids to about 165 aminoacids, about 95 amino acids to about 160 amino acids, about 95 aminoacids to about 155 amino acids, about 95 amino acids to about 150 aminoacids, about 95 amino acids to about 145 amino acids, about 95 aminoacids to about 140 amino acids, about 95 amino acids to about 135 aminoacids, about 95 amino acids to about 130 amino acids, about 95 aminoacids to about 125 amino acids, about 95 amino acids to about 120 aminoacids, about 95 amino acids to about 115 amino acids, about 95 aminoacids to about 110 amino acids, about 95 amino acids to about 105 aminoacids, about 95 amino acids to about 100 amino acids, about 100 aminoacids to about 1000 amino acids, about 100 amino acids to about 950amino acids, about 100 amino acids to about 900 amino acids, about 100amino acids to about 850 amino acids, about 100 amino acids to about 800amino acids, about 100 amino acids to about 750 amino acids, about 100amino acids to about 700 amino acids, about 100 amino acids to about 650amino acids, about 100 amino acids to about 600 amino acids, about 100amino acids to about 550 amino acids, about 100 amino acids to about 500amino acids, about 100 amino acids to about 450 amino acids, about 100amino acids to about 400 amino acids, about 100 amino acids to about 350amino acids, about 100 amino acids to about 300 amino acids, about 100amino acids to about 280 amino acids, about 100 amino acids to about 260amino acids, about 100 amino acids to about 240 amino acids, about 100amino acids to about 220 amino acids, about 100 amino acids to about 200amino acids, about 100 amino acids to about 195 amino acids, about 100amino acids to about 190 amino acids, about 100 amino acids to about 185amino acids, about 100 amino acids to about 180 amino acids, about 100amino acids to about 175 amino acids, about 100 amino acids to about 170amino acids, about 100 amino acids to about 165 amino acids, about 100amino acids to about 160 amino acids, about 100 amino acids to about 155amino acids, about 100 amino acids to about 150 amino acids, about 100amino acids to about 145 amino acids, about 100 amino acids to about 140amino acids, about 100 amino acids to about 135 amino acids, about 100amino acids to about 130 amino acids, about 100 amino acids to about 125amino acids, about 100 amino acids to about 120 amino acids, about 100amino acids to about 115 amino acids, about 100 amino acids to about 110amino acids, about 100 amino acids to about 105 amino acids, about 105amino acids to about 1000 amino acids, about 105 amino acids to about950 amino acids, about 105 amino acids to about 900 amino acids, about105 amino acids to about 850 amino acids, about 105 amino acids to about800 amino acids, about 105 amino acids to about 750 amino acids, about105 amino acids to about 700 amino acids, about 105 amino acids to about650 amino acids, about 105 amino acids to about 600 amino acids, about105 amino acids to about 550 amino acids, about 105 amino acids to about500 amino acids, about 105 amino acids to about 450 amino acids, about105 amino acids to about 400 amino acids, about 105 amino acids to about350 amino acids, about 105 amino acids to about 300 amino acids, about105 amino acids to about 280 amino acids, about 105 amino acids to about260 amino acids, about 105 amino acids to about 240 amino acids, about105 amino acids to about 220 amino acids, about 105 amino acids to about200 amino acids, about 105 amino acids to about 195 amino acids, about105 amino acids to about 190 amino acids, about 105 amino acids to about185 amino acids, about 105 amino acids to about 180 amino acids, about105 amino acids to about 175 amino acids, about 105 amino acids to about170 amino acids, about 105 amino acids to about 165 amino acids, about105 amino acids to about 160 amino acids, about 105 amino acids to about155 amino acids, about 105 amino acids to about 150 amino acids, about105 amino acids to about 145 amino acids, about 105 amino acids to about140 amino acids, about 105 amino acids to about 135 amino acids, about105 amino acids to about 130 amino acids, about 105 amino acids to about125 amino acids, about 105 amino acids to about 120 amino acids, about105 amino acids to about 115 amino acids, about 105 amino acids to about110 amino acids, about 110 amino acids to about 1000 amino acids, about110 amino acids to about 950 amino acids, about 110 amino acids to about900 amino acids, about 110 amino acids to about 850 amino acids, about110 amino acids to about 800 amino acids, about 110 amino acids to about750 amino acids, about 110 amino acids to about 700 amino acids, about110 amino acids to about 650 amino acids, about 110 amino acids to about600 amino acids, about 110 amino acids to about 550 amino acids, about110 amino acids to about 500 amino acids, about 110 amino acids to about450 amino acids, about 110 amino acids to about 400 amino acids, about110 amino acids to about 350 amino acids, about 110 amino acids to about300 amino acids, about 110 amino acids to about 280 amino acids, about110 amino acids to about 260 amino acids, about 110 amino acids to about240 amino acids, about 110 amino acids to about 220 amino acids, about110 amino acids to about 200 amino acids, about 110 amino acids to about195 amino acids, about 110 amino acids to about 190 amino acids, about110 amino acids to about 185 amino acids, about 110 amino acids to about180 amino acids, about 110 amino acids to about 175 amino acids, about110 amino acids to about 170 amino acids, about 110 amino acids to about165 amino acids, about 110 amino acids to about 160 amino acids, about110 amino acids to about 155 amino acids, about 110 amino acids to about150 amino acids, about 110 amino acids to about 145 amino acids, about110 amino acids to about 140 amino acids, about 110 amino acids to about135 amino acids, about 110 amino acids to about 130 amino acids, about110 amino acids to about 125 amino acids, about 110 amino acids to about120 amino acids, about 110 amino acids to about 115 amino acids, about115 amino acids to about 1000 amino acids, about 115 amino acids toabout 950 amino acids, about 115 amino acids to about 900 amino acids,about 115 amino acids to about 850 amino acids, about 115 amino acids toabout 800 amino acids, about 115 amino acids to about 750 amino acids,about 115 amino acids to about 700 amino acids, about 115 amino acids toabout 650 amino acids, about 115 amino acids to about 600 amino acids,about 115 amino acids to about 550 amino acids, about 115 amino acids toabout 500 amino acids, about 115 amino acids to about 450 amino acids,about 115 amino acids to about 400 amino acids, about 115 amino acids toabout 350 amino acids, about 115 amino acids to about 300 amino acids,about 115 amino acids to about 280 amino acids, about 115 amino acids toabout 260 amino acids, about 115 amino acids to about 240 amino acids,about 115 amino acids to about 220 amino acids, about 115 amino acids toabout 200 amino acids, about 115 amino acids to about 195 amino acids,about 115 amino acids to about 190 amino acids, about 115 amino acids toabout 185 amino acids, about 115 amino acids to about 180 amino acids,about 115 amino acids to about 175 amino acids, about 115 amino acids toabout 170 amino acids, about 115 amino acids to about 165 amino acids,about 115 amino acids to about 160 amino acids, about 115 amino acids toabout 155 amino acids, about 115 amino acids to about 150 amino acids,about 115 amino acids to about 145 amino acids, about 115 amino acids toabout 140 amino acids, about 115 amino acids to about 135 amino acids,about 115 amino acids to about 130 amino acids, about 115 amino acids toabout 125 amino acids, about 115 amino acids to about 120 amino acids,about 120 amino acids to about 1000 amino acids, about 120 amino acidsto about 950 amino acids, about 120 amino acids to about 900 aminoacids, about 120 amino acids to about 850 amino acids, about 120 aminoacids to about 800 amino acids, about 120 amino acids to about 750 aminoacids, about 120 amino acids to about 700 amino acids, about 120 aminoacids to about 650 amino acids, about 120 amino acids to about 600 aminoacids, about 120 amino acids to about 550 amino acids, about 120 aminoacids to about 500 amino acids, about 120 amino acids to about 450 aminoacids, about 120 amino acids to about 400 amino acids, about 120 aminoacids to about 350 amino acids, about 120 amino acids to about 300 aminoacids, about 120 amino acids to about 280 amino acids, about 120 aminoacids to about 260 amino acids, about 120 amino acids to about 240 aminoacids, about 120 amino acids to about 220 amino acids, about 120 aminoacids to about 200 amino acids, about 120 amino acids to about 195 aminoacids, about 120 amino acids to about 190 amino acids, about 120 aminoacids to about 185 amino acids, about 120 amino acids to about 180 aminoacids, about 120 amino acids to about 175 amino acids, about 120 aminoacids to about 170 amino acids, about 120 amino acids to about 165 aminoacids, about 120 amino acids to about 160 amino acids, about 120 aminoacids to about 155 amino acids, about 120 amino acids to about 150 aminoacids, about 120 amino acids to about 145 amino acids, about 120 aminoacids to about 140 amino acids, about 120 amino acids to about 135 aminoacids, about 120 amino acids to about 130 amino acids, about 120 aminoacids to about 125 amino acids, about 125 amino acids to about 1000amino acids, about 125 amino acids to about 950 amino acids, about 125amino acids to about 900 amino acids, about 125 amino acids to about 850amino acids, about 125 amino acids to about 800 amino acids, about 125amino acids to about 750 amino acids, about 125 amino acids to about 700amino acids, about 125 amino acids to about 650 amino acids, about 125amino acids to about 600 amino acids, about 125 amino acids to about 550amino acids, about 125 amino acids to about 500 amino acids, about 125amino acids to about 450 amino acids, about 125 amino acids to about 400amino acids, about 125 amino acids to about 350 amino acids, about 125amino acids to about 300 amino acids, about 125 amino acids to about 280amino acids, about 125 amino acids to about 260 amino acids, about 125amino acids to about 240 amino acids, about 125 amino acids to about 220amino acids, about 125 amino acids to about 200 amino acids, about 125amino acids to about 195 amino acids, about 125 amino acids to about 190amino acids, about 125 amino acids to about 185 amino acids, about 125amino acids to about 180 amino acids, about 125 amino acids to about 175amino acids, about 125 amino acids to about 170 amino acids, about 125amino acids to about 165 amino acids, about 125 amino acids to about 160amino acids, about 125 amino acids to about 155 amino acids, about 125amino acids to about 150 amino acids, about 125 amino acids to about 145amino acids, about 125 amino acids to about 140 amino acids, about 125amino acids to about 135 amino acids, about 125 amino acids to about 130amino acids, about 130 amino acids to about 1000 amino acids, about 130amino acids to about 950 amino acids, about 130 amino acids to about 900amino acids, about 130 amino acids to about 850 amino acids, about 130amino acids to about 800 amino acids, about 130 amino acids to about 750amino acids, about 130 amino acids to about 700 amino acids, about 130amino acids to about 650 amino acids, about 130 amino acids to about 600amino acids, about 130 amino acids to about 550 amino acids, about 130amino acids to about 500 amino acids, about 130 amino acids to about 450amino acids, about 130 amino acids to about 400 amino acids, about 130amino acids to about 350 amino acids, about 130 amino acids to about 300amino acids, about 130 amino acids to about 280 amino acids, about 130amino acids to about 260 amino acids, about 130 amino acids to about 240amino acids, about 130 amino acids to about 220 amino acids, about 130amino acids to about 200 amino acids, about 130 amino acids to about 195amino acids, about 130 amino acids to about 190 amino acids, about 130amino acids to about 185 amino acids, about 130 amino acids to about 180amino acids, about 130 amino acids to about 175 amino acids, about 130amino acids to about 170 amino acids, about 130 amino acids to about 165amino acids, about 130 amino acids to about 160 amino acids, about 130amino acids to about 155 amino acids, about 130 amino acids to about 150amino acids, about 130 amino acids to about 145 amino acids, about 130amino acids to about 140 amino acids, about 130 amino acids to about 135amino acids, about 135 amino acids to about 1000 amino acids, about 135amino acids to about 950 amino acids, about 135 amino acids to about 900amino acids, about 135 amino acids to about 850 amino acids, about 135amino acids to about 800 amino acids, about 135 amino acids to about 750amino acids, about 135 amino acids to about 700 amino acids, about 135amino acids to about 650 amino acids, about 135 amino acids to about 600amino acids, about 135 amino acids to about 550 amino acids, about 135amino acids to about 500 amino acids, about 135 amino acids to about 450amino acids, about 135 amino acids to about 400 amino acids, about 135amino acids to about 350 amino acids, about 135 amino acids to about 300amino acids, about 135 amino acids to about 280 amino acids, about 135amino acids to about 260 amino acids, about 135 amino acids to about 240amino acids, about 135 amino acids to about 220 amino acids, about 135amino acids to about 200 amino acids, about 135 amino acids to about 195amino acids, about 135 amino acids to about 190 amino acids, about 135amino acids to about 185 amino acids, about 135 amino acids to about 180amino acids, about 135 amino acids to about 175 amino acids, about 135amino acids to about 170 amino acids, about 135 amino acids to about 165amino acids, about 135 amino acids to about 160 amino acids, about 135amino acids to about 155 amino acids, about 135 amino acids to about 150amino acids, about 135 amino acids to about 145 amino acids, about 135amino acids to about 140 amino acids, about 140 amino acids to about1000 amino acids, about 140 amino acids to about 950 amino acids, about140 amino acids to about 900 amino acids, about 140 amino acids to about850 amino acids, about 140 amino acids to about 800 amino acids, about140 amino acids to about 750 amino acids, about 140 amino acids to about700 amino acids, about 140 amino acids to about 650 amino acids, about140 amino acids to about 600 amino acids, about 140 amino acids to about550 amino acids, about 140 amino acids to about 500 amino acids, about140 amino acids to about 450 amino acids, about 140 amino acids to about400 amino acids, about 140 amino acids to about 350 amino acids, about140 amino acids to about 300 amino acids, about 140 amino acids to about280 amino acids, about 140 amino acids to about 260 amino acids, about140 amino acids to about 240 amino acids, about 140 amino acids to about220 amino acids, about 140 amino acids to about 200 amino acids, about140 amino acids to about 195 amino acids, about 140 amino acids to about190 amino acids, about 140 amino acids to about 185 amino acids, about140 amino acids to about 180 amino acids, about 140 amino acids to about175 amino acids, about 140 amino acids to about 170 amino acids, about140 amino acids to about 165 amino acids, about 140 amino acids to about160 amino acids, about 140 amino acids to about 155 amino acids, about140 amino acids to about 150 amino acids, about 140 amino acids to about145 amino acids, about 145 amino acids to about 1000 amino acids, about145 amino acids to about 950 amino acids, about 145 amino acids to about900 amino acids, about 145 amino acids to about 850 amino acids, about145 amino acids to about 800 amino acids, about 145 amino acids to about750 amino acids, about 145 amino acids to about 700 amino acids, about145 amino acids to about 650 amino acids, about 145 amino acids to about600 amino acids, about 145 amino acids to about 550 amino acids, about145 amino acids to about 500 amino acids, about 145 amino acids to about450 amino acids, about 145 amino acids to about 400 amino acids, about145 amino acids to about 350 amino acids, about 145 amino acids to about300 amino acids, about 145 amino acids to about 280 amino acids, about145 amino acids to about 260 amino acids, about 145 amino acids to about240 amino acids, about 145 amino acids to about 220 amino acids, about145 amino acids to about 200 amino acids, about 145 amino acids to about195 amino acids, about 145 amino acids to about 190 amino acids, about145 amino acids to about 185 amino acids, about 145 amino acids to about180 amino acids, about 145 amino acids to about 175 amino acids, about145 amino acids to about 170 amino acids, about 145 amino acids to about165 amino acids, about 145 amino acids to about 160 amino acids, about145 amino acids to about 155 amino acids, about 145 amino acids to about150 amino acids, about 150 amino acids to about 1000 amino acids, about150 amino acids to about 950 amino acids, about 150 amino acids to about900 amino acids, about 150 amino acids to about 850 amino acids, about150 amino acids to about 800 amino acids, about 150 amino acids to about750 amino acids, about 150 amino acids to about 700 amino acids, about150 amino acids to about 650 amino acids, about 150 amino acids to about600 amino acids, about 150 amino acids to about 550 amino acids, about150 amino acids to about 500 amino acids, about 150 amino acids to about450 amino acids, about 150 amino acids to about 400 amino acids, about150 amino acids to about 350 amino acids, about 150 amino acids to about300 amino acids, about 150 amino acids to about 280 amino acids, about150 amino acids to about 260 amino acids, about 150 amino acids to about240 amino acids, about 150 amino acids to about 220 amino acids, about150 amino acids to about 200 amino acids, about 150 amino acids to about195 amino acids, about 150 amino acids to about 190 amino acids, about150 amino acids to about 185 amino acids, about 150 amino acids to about180 amino acids, about 150 amino acids to about 175 amino acids, about150 amino acids to about 170 amino acids, about 150 amino acids to about165 amino acids, about 150 amino acids to about 160 amino acids, about150 amino acids to about 155 amino acids, about 155 amino acids to about1000 amino acids, about 155 amino acids to about 950 amino acids, about155 amino acids to about 900 amino acids, about 155 amino acids to about850 amino acids, about 155 amino acids to about 800 amino acids, about155 amino acids to about 750 amino acids, about 155 amino acids to about700 amino acids, about 155 amino acids to about 650 amino acids, about155 amino acids to about 600 amino acids, about 155 amino acids to about550 amino acids, about 155 amino acids to about 500 amino acids, about155 amino acids to about 450 amino acids, about 155 amino acids to about400 amino acids, about 155 amino acids to about 350 amino acids, about155 amino acids to about 300 amino acids, about 155 amino acids to about280 amino acids, about 155 amino acids to about 260 amino acids, about155 amino acids to about 240 amino acids, about 155 amino acids to about220 amino acids, about 155 amino acids to about 200 amino acids, about155 amino acids to about 195 amino acids, about 155 amino acids to about190 amino acids, about 155 amino acids to about 185 amino acids, about155 amino acids to about 180 amino acids, about 155 amino acids to about175 amino acids, about 155 amino acids to about 170 amino acids, about155 amino acids to about 165 amino acids, about 155 amino acids to about160 amino acids, about 160 amino acids to about 1000 amino acids, about160 amino acids to about 950 amino acids, about 160 amino acids to about900 amino acids, about 160 amino acids to about 850 amino acids, about160 amino acids to about 800 amino acids, about 160 amino acids to about750 amino acids, about 160 amino acids to about 700 amino acids, about160 amino acids to about 650 amino acids, about 160 amino acids to about600 amino acids, about 160 amino acids to about 550 amino acids, about160 amino acids to about 500 amino acids, about 160 amino acids to about450 amino acids, about 160 amino acids to about 400 amino acids, about160 amino acids to about 350 amino acids, about 160 amino acids to about300 amino acids, about 160 amino acids to about 280 amino acids, about160 amino acids to about 260 amino acids, about 160 amino acids to about240 amino acids, about 160 amino acids to about 220 amino acids, about160 amino acids to about 200 amino acids, about 160 amino acids to about195 amino acids, about 160 amino acids to about 190 amino acids, about160 amino acids to about 185 amino acids, about 160 amino acids to about180 amino acids, about 160 amino acids to about 175 amino acids, about160 amino acids to about 170 amino acids, about 160 amino acids to about165 amino acids, about 165 amino acids to about 1000 amino acids, about165 amino acids to about 950 amino acids, about 165 amino acids to about900 amino acids, about 165 amino acids to about 850 amino acids, about165 amino acids to about 800 amino acids, about 165 amino acids to about750 amino acids, about 165 amino acids to about 700 amino acids, about165 amino acids to about 650 amino acids, about 165 amino acids to about600 amino acids, about 165 amino acids to about 550 amino acids, about165 amino acids to about 500 amino acids, about 165 amino acids to about450 amino acids, about 165 amino acids to about 400 amino acids, about165 amino acids to about 350 amino acids, about 165 amino acids to about300 amino acids, about 165 amino acids to about 280 amino acids, about165 amino acids to about 260 amino acids, about 165 amino acids to about240 amino acids, about 165 amino acids to about 220 amino acids, about165 amino acids to about 200 amino acids, about 165 amino acids to about195 amino acids, about 165 amino acids to about 190 amino acids, about165 amino acids to about 185 amino acids, about 165 amino acids to about180 amino acids, about 165 amino acids to about 175 amino acids, about165 amino acids to about 170 amino acids, about 170 amino acids to about1000 amino acids, about 170 amino acids to about 950 amino acids, about170 amino acids to about 900 amino acids, about 170 amino acids to about850 amino acids, about 170 amino acids to about 800 amino acids, about170 amino acids to about 750 amino acids, about 170 amino acids to about700 amino acids, about 170 amino acids to about 650 amino acids, about170 amino acids to about 600 amino acids, about 170 amino acids to about550 amino acids, about 170 amino acids to about 500 amino acids, about170 amino acids to about 450 amino acids, about 170 amino acids to about400 amino acids, about 170 amino acids to about 350 amino acids, about170 amino acids to about 300 amino acids, about 170 amino acids to about280 amino acids, about 170 amino acids to about 260 amino acids, about170 amino acids to about 240 amino acids, about 170 amino acids to about220 amino acids, about 170 amino acids to about 200 amino acids, about170 amino acids to about 195 amino acids, about 170 amino acids to about190 amino acids, about 170 amino acids to about 185 amino acids, about170 amino acids to about 180 amino acids, about 170 amino acids to about175 amino acids, about 175 amino acids to about 1000 amino acids, about175 amino acids to about 950 amino acids, about 175 amino acids to about900 amino acids, about 175 amino acids to about 850 amino acids, about175 amino acids to about 800 amino acids, about 175 amino acids to about750 amino acids, about 175 amino acids to about 700 amino acids, about175 amino acids to about 650 amino acids, about 175 amino acids to about600 amino acids, about 175 amino acids to about 550 amino acids, about175 amino acids to about 500 amino acids, about 175 amino acids to about450 amino acids, about 175 amino acids to about 400 amino acids, about175 amino acids to about 350 amino acids, about 175 amino acids to about300 amino acids, about 175 amino acids to about 280 amino acids, about175 amino acids to about 260 amino acids, about 175 amino acids to about240 amino acids, about 175 amino acids to about 220 amino acids, about175 amino acids to about 200 amino acids, about 175 amino acids to about195 amino acids, about 175 amino acids to about 190 amino acids, about175 amino acids to about 185 amino acids, about 175 amino acids to about180 amino acids, about 180 amino acids to about 1000 amino acids, about180 amino acids to about 950 amino acids, about 180 amino acids to about900 amino acids, about 180 amino acids to about 850 amino acids, about180 amino acids to about 800 amino acids, about 180 amino acids to about750 amino acids, about 180 amino acids to about 700 amino acids, about180 amino acids to about 650 amino acids, about 180 amino acids to about600 amino acids, about 180 amino acids to about 550 amino acids, about180 amino acids to about 500 amino acids, about 180 amino acids to about450 amino acids, about 180 amino acids to about 400 amino acids, about180 amino acids to about 350 amino acids, about 180 amino acids to about300 amino acids, about 180 amino acids to about 280 amino acids, about180 amino acids to about 260 amino acids, about 180 amino acids to about240 amino acids, about 180 amino acids to about 220 amino acids, about180 amino acids to about 200 amino acids, about 180 amino acids to about195 amino acids, about 180 amino acids to about 190 amino acids, about180 amino acids to about 185 amino acids, about 185 amino acids to about1000 amino acids, about 185 amino acids to about 950 amino acids, about185 amino acids to about 900 amino acids, about 185 amino acids to about850 amino acids, about 185 amino acids to about 800 amino acids, about185 amino acids to about 750 amino acids, about 185 amino acids to about700 amino acids, about 185 amino acids to about 650 amino acids, about185 amino acids to about 600 amino acids, about 185 amino acids to about550 amino acids, about 185 amino acids to about 500 amino acids, about185 amino acids to about 450 amino acids, about 185 amino acids to about400 amino acids, about 185 amino acids to about 350 amino acids, about185 amino acids to about 300 amino acids, about 185 amino acids to about280 amino acids, about 185 amino acids to about 260 amino acids, about185 amino acids to about 240 amino acids, about 185 amino acids to about220 amino acids, about 185 amino acids to about 200 amino acids, about185 amino acids to about 195 amino acids, about 185 amino acids to about190 amino acids, about 190 amino acids to about 1000 amino acids, about190 amino acids to about 950 amino acids, about 190 amino acids to about900 amino acids, about 190 amino acids to about 850 amino acids, about190 amino acids to about 800 amino acids, about 190 amino acids to about750 amino acids, about 190 amino acids to about 700 amino acids, about190 amino acids to about 650 amino acids, about 190 amino acids to about600 amino acids, about 190 amino acids to about 550 amino acids, about190 amino acids to about 500 amino acids, about 190 amino acids to about450 amino acids, about 190 amino acids to about 400 amino acids, about190 amino acids to about 350 amino acids, about 190 amino acids to about300 amino acids, about 190 amino acids to about 280 amino acids, about190 amino acids to about 260 amino acids, about 190 amino acids to about240 amino acids, about 190 amino acids to about 220 amino acids, about190 amino acids to about 200 amino acids, about 190 amino acids to about195 amino acids, about 195 amino acids to about 1000 amino acids, about195 amino acids to about 950 amino acids, about 195 amino acids to about900 amino acids, about 195 amino acids to about 850 amino acids, about195 amino acids to about 800 amino acids, about 195 amino acids to about750 amino acids, about 195 amino acids to about 700 amino acids, about195 amino acids to about 650 amino acids, about 195 amino acids to about600 amino acids, about 195 amino acids to about 550 amino acids, about195 amino acids to about 500 amino acids, about 195 amino acids to about450 amino acids, about 195 amino acids to about 400 amino acids, about195 amino acids to about 350 amino acids, about 195 amino acids to about300 amino acids, about 195 amino acids to about 280 amino acids, about195 amino acids to about 260 amino acids, about 195 amino acids to about240 amino acids, about 195 amino acids to about 220 amino acids, about195 amino acids to about 200 amino acids, about 200 amino acids to about1000 amino acids, about 200 amino acids to about 950 amino acids, about200 amino acids to about 900 amino acids, about 200 amino acids to about850 amino acids, about 200 amino acids to about 800 amino acids, about200 amino acids to about 750 amino acids, about 200 amino acids to about700 amino acids, about 200 amino acids to about 650 amino acids, about200 amino acids to about 600 amino acids, about 200 amino acids to about550 amino acids, about 200 amino acids to about 500 amino acids, about200 amino acids to about 450 amino acids, about 200 amino acids to about400 amino acids, about 200 amino acids to about 350 amino acids, about200 amino acids to about 300 amino acids, about 200 amino acids to about280 amino acids, about 200 amino acids to about 260 amino acids, about200 amino acids to about 240 amino acids, about 200 amino acids to about220 amino acids, about 220 amino acids to about 1000 amino acids, about220 amino acids to about 950 amino acids, about 220 amino acids to about900 amino acids, about 220 amino acids to about 850 amino acids, about220 amino acids to about 800 amino acids, about 220 amino acids to about750 amino acids, about 220 amino acids to about 700 amino acids, about220 amino acids to about 650 amino acids, about 220 amino acids to about600 amino acids, about 220 amino acids to about 550 amino acids, about220 amino acids to about 500 amino acids, about 220 amino acids to about450 amino acids, about 220 amino acids to about 400 amino acids, about220 amino acids to about 350 amino acids, about 220 amino acids to about300 amino acids, about 220 amino acids to about 280 amino acids, about220 amino acids to about 260 amino acids, about 220 amino acids to about240 amino acids, about 240 amino acids to about 1000 amino acids, about240 amino acids to about 950 amino acids, about 240 amino acids to about900 amino acids, about 240 amino acids to about 850 amino acids, about240 amino acids to about 800 amino acids, about 240 amino acids to about750 amino acids, about 240 amino acids to about 700 amino acids, about240 amino acids to about 650 amino acids, about 240 amino acids to about600 amino acids, about 240 amino acids to about 550 amino acids, about240 amino acids to about 500 amino acids, about 240 amino acids to about450 amino acids, about 240 amino acids to about 400 amino acids, about240 amino acids to about 350 amino acids, about 240 amino acids to about300 amino acids, about 240 amino acids to about 280 amino acids, about240 amino acids to about 260 amino acids, about 260 amino acids to about1000 amino acids, about 260 amino acids to about 950 amino acids, about260 amino acids to about 900 amino acids, about 260 amino acids to about850 amino acids, about 260 amino acids to about 800 amino acids, about260 amino acids to about 750 amino acids, about 260 amino acids to about700 amino acids, about 260 amino acids to about 650 amino acids, about260 amino acids to about 600 amino acids, about 260 amino acids to about550 amino acids, about 260 amino acids to about 500 amino acids, about260 amino acids to about 450 amino acids, about 260 amino acids to about400 amino acids, about 260 amino acids to about 350 amino acids, about260 amino acids to about 300 amino acids, about 260 amino acids to about280 amino acids, about 280 amino acids to about 1000 amino acids, about280 amino acids to about 950 amino acids, about 280 amino acids to about900 amino acids, about 280 amino acids to about 850 amino acids, about280 amino acids to about 800 amino acids, about 280 amino acids to about750 amino acids, about 280 amino acids to about 700 amino acids, about280 amino acids to about 650 amino acids, about 280 amino acids to about600 amino acids, about 280 amino acids to about 550 amino acids, about280 amino acids to about 500 amino acids, about 280 amino acids to about450 amino acids, about 280 amino acids to about 400 amino acids, about280 amino acids to about 350 amino acids, about 280 amino acids to about300 amino acids, about 300 amino acids to about 1000 amino acids, about300 amino acids to about 950 amino acids, about 300 amino acids to about900 amino acids, about 300 amino acids to about 850 amino acids, about300 amino acids to about 800 amino acids, about 300 amino acids to about750 amino acids, about 300 amino acids to about 700 amino acids, about300 amino acids to about 650 amino acids, about 300 amino acids to about600 amino acids, about 300 amino acids to about 550 amino acids, about300 amino acids to about 500 amino acids, about 300 amino acids to about450 amino acids, about 300 amino acids to about 400 amino acids, about300 amino acids to about 350 amino acids, about 350 amino acids to about1000 amino acids, about 350 amino acids to about 950 amino acids, about350 amino acids to about 900 amino acids, about 350 amino acids to about850 amino acids, about 350 amino acids to about 800 amino acids, about350 amino acids to about 750 amino acids, about 350 amino acids to about700 amino acids, about 350 amino acids to about 650 amino acids, about350 amino acids to about 600 amino acids, about 350 amino acids to about550 amino acids, about 350 amino acids to about 500 amino acids, about350 amino acids to about 450 amino acids, about 350 amino acids to about400 amino acids, about 400 amino acids to about 1000 amino acids, about400 amino acids to about 950 amino acids, about 400 amino acids to about900 amino acids, about 400 amino acids to about 850 amino acids, about400 amino acids to about 800 amino acids, about 400 amino acids to about750 amino acids, about 400 amino acids to about 700 amino acids, about400 amino acids to about 650 amino acids, about 400 amino acids to about600 amino acids, about 400 amino acids to about 550 amino acids, about400 amino acids to about 500 amino acids, about 400 amino acids to about450 amino acids, about 450 amino acids to about 1000 amino acids, about450 amino acids to about 950 amino acids, about 450 amino acids to about900 amino acids, about 450 amino acids to about 850 amino acids, about450 amino acids to about 800 amino acids, about 450 amino acids to about750 amino acids, about 450 amino acids to about 700 amino acids, about450 amino acids to about 650 amino acids, about 450 amino acids to about600 amino acids, about 450 amino acids to about 550 amino acids, about450 amino acids to about 500 amino acids, about 500 amino acids to about1000 amino acids, about 500 amino acids to about 950 amino acids, about500 amino acids to about 900 amino acids, about 500 amino acids to about850 amino acids, about 500 amino acids to about 800 amino acids, about500 amino acids to about 750 amino acids, about 500 amino acids to about700 amino acids, about 500 amino acids to about 650 amino acids, about500 amino acids to about 600 amino acids, about 500 amino acids to about550 amino acids, about 550 amino acids to about 1000 amino acids, about550 amino acids to about 950 amino acids, about 550 amino acids to about900 amino acids, about 550 amino acids to about 850 amino acids, about550 amino acids to about 800 amino acids, about 550 amino acids to about750 amino acids, about 550 amino acids to about 700 amino acids, about550 amino acids to about 650 amino acids, about 550 amino acids to about600 amino acids, about 600 amino acids to about 1000 amino acids, about600 amino acids to about 950 amino acids, about 600 amino acids to about900 amino acids, about 600 amino acids to about 850 amino acids, about600 amino acids to about 800 amino acids, about 600 amino acids to about750 amino acids, about 600 amino acids to about 700 amino acids, about600 amino acids to about 650 amino acids, about 650 amino acids to about1000 amino acids, about 650 amino acids to about 950 amino acids, about650 amino acids to about 900 amino acids, about 650 amino acids to about850 amino acids, about 650 amino acids to about 800 amino acids, about650 amino acids to about 750 amino acids, about 650 amino acids to about700 amino acids, about 700 amino acids to about 1000 amino acids, about700 amino acids to about 950 amino acids, about 700 amino acids to about900 amino acids, about 700 amino acids to about 850 amino acids, about700 amino acids to about 800 amino acids, about 700 amino acids to about750 amino acids, about 750 amino acids to about 1000 amino acids, about750 amino acids to about 950 amino acids, about 750 amino acids to about900 amino acids, about 750 amino acids to about 850 amino acids, about750 amino acids to about 800 amino acids, about 800 amino acids to about1000 amino acids, about 800 amino acids to about 950 amino acids, about800 amino acids to about 900 amino acids, about 800 amino acids to about850 amino acids, about 850 amino acids to about 1000 amino acids, about850 amino acids to about 950 amino acids, about 850 amino acids to about900 amino acids, about 900 amino acids to about 1000 amino acids, about900 amino acids to about 950 amino acids, or about 950 amino acids toabout 1000 amino acids.

Any of the target-binding domains described herein can bind to itstarget with a dissociation equilibrium constant (K_(D)) of less than1×10⁻⁷ M, less than 1×10⁻⁸M, less than 1×10⁻⁹M, less than 1×10⁻¹⁰ M,less than 1×10⁻¹¹M, less than 1×10⁻¹²M, or less than 1×10⁻¹³ M. In someembodiments, the antigen-binding protein construct provided herein canbind to an identifying antigen with a K_(D) of about 1×10⁻³M to about1×10⁻⁵ M, about 1×10⁻⁷ M to about 1×10⁻⁶ M, about 1×10⁻⁵M to about1×10⁻⁷ M, about 1×10⁻⁶M to about 1×10⁻⁸M, about 1×10⁻⁷ M to about1×10⁻⁹M, about 1×10⁻⁸M to about 1×10⁻¹⁰ M, or about 1×10⁻⁹M to about1×10⁻¹¹M (inclusive).

Any of the target-binding domains described herein can bind to itstarget with a K_(D) of between about 1 pM to about 30 nM (e.g., about 1pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM,about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about2 nM, about 1 pM to about 1 nM, about 1 pM to about 950 pM, about 1 pMto about 900 pM, about 1 pM to about 850 pM, about 1 pM to about 800 pM,about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM toabout 650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM,about 1 pM to about 500 pM, about 1 pM to about 450 pM, about 1 pM toabout 400 pM, about 1 pM to about 350 pM, about 1 pM to about 300 pM,about 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM toabout 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM,about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM toabout 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM toabout 3 pM, about 1 pM to about 2 pM, about 2 pM to about 30 nM, about 2pM to about 25 nM, about 2 pM to about 20 nM, about 2 pM to about 15 nM,about 2 pM to about 10 nM, about 2 pM to about 5 nM, about 2 pM to about2 nM, about 2 pM to about 1 nM, about 2 pM to about 950 pM, about 2 pMto about 900 pM, about 2 pM to about 850 pM, about 2 pM to about 800 pM,about 2 pM to about 750 pM, about 2 pM to about 700 pM, about 2 pM toabout 650 pM, about 2 pM to about 600 pM, about 2 pM to about 550 pM,about 2 pM to about 500 pM, about 2 pM to about 450 pM, about 2 pM toabout 400 pM, about 2 pM to about 350 pM, about 2 pM to about 300 pM,about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2 pM toabout 150 pM, about 2 pM to about 100 pM, about 2 pM to about 90 pM,about 2 pM to about 80 pM, about 2 pM to about 70 pM, about 2 pM toabout 60 pM, about 2 pM to about 50 pM, about 2 pM to about 40 pM, about2 pM to about 30 pM, about 2 pM to about 20 pM, about 2 pM to about 10pM, about 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM toabout 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about5 pM to about 20 nM, about 5 pM to about 15 nM, about 5 pM to about 10nM, about 5 pM to about 5 nM, about 5 pM to about 2 nM, about 5 pM toabout 1 nM, about 5 pM to about 950 pM, about 5 pM to about 900 pM,about 5 pM to about 850 pM, about 5 pM to about 800 pM, about 5 pM toabout 750 pM, about 5 pM to about 700 pM, about 5 pM to about 650 pM,about 5 pM to about 600 pM, about 5 pM to about 550 pM, about 5 pM toabout 500 pM, about 5 pM to about 450 pM, about 5 pM to about 400 pM,about 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM toabout 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM,about 5 pM to about 100 pM, about 5 pM to about 90 pM, about 5 pM toabout 80 pM, about 5 pM to about 70 pM, about 5 pM to about 60 pM, about5 pM to about 50 pM, about 5 pM to about 40 pM, about 5 pM to about 30pM, about 5 pM to about 20 pM, about 5 pM to about 10 pM, about 10 pM toabout 30 nM, about 10 pM to about 25 nM, about 10 pM to about 20 nM,about 10 pM to about 15 nM, about 10 pM to about 10 nM, about 10 pM toabout 5 nM, about 10 pM to about 2 nM, about 10 pM to about 1 nM, about10 pM to about 950 pM, about 10 pM to about 900 pM, about 10 pM to about850 pM, about 10 pM to about 800 pM, about 10 pM to about 750 pM, about10 pM to about 700 pM, about 10 pM to about 650 pM, about 10 pM to about600 pM, about 10 pM to about 550 pM, about 10 pM to about 500 pM, about10 pM to about 450 pM, about 10 pM to about 400 pM, about 10 pM to about350 pM, about 10 pM to about 300 pM, about 10 pM to about 250 pM, about10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about100 pM, about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10pM to about 70 pM, about 10 pM to about 60 pM, about 10 pM to about 50pM, about 10 pM to about 40 pM, about 10 pM to about 30 pM, about 10 pMto about 20 pM, about 15 pM to about 30 nM, about 15 pM to about 25 nM,about 15 pM to about 20 nM, about 15 pM to about 15 nM, about 15 pM toabout 10 nM, about 15 pM to about 5 nM, about 15 pM to about 2 nM, about15 pM to about 1 nM, about 15 pM to about 950 pM, about 15 pM to about900 pM, about 15 pM to about 850 pM, about 15 pM to about 800 pM, about15 pM to about 750 pM, about 15 pM to about 700 pM, about 15 pM to about650 pM, about 15 pM to about 600 pM, about 15 pM to about 550 pM, about15 pM to about 500 pM, about 15 pM to about 450 pM, about 15 pM to about400 pM, about 15 pM to about 350 pM, about 15 pM to about 300 pM, about15 pM to about 250 pM, about 15 pM to about 200 pM, about 15 pM to about150 pM, about 15 pM to about 100 pM, about 15 pM to about 90 pM, about15 pM to about 80 pM, about 15 pM to about 70 pM, about 15 pM to about60 pM, about 15 pM to about 50 pM, about 15 pM to about 40 pM, about 15pM to about 30 pM, about 15 pM to about 20 pM, about 20 pM to about 30nM, about 20 pM to about 25 nM, about 20 pM to about 20 nM, about 20 pMto about 15 nM, about 20 pM to about 10 nM, about 20 pM to about 5 nM,about 20 pM to about 2 nM, about 20 pM to about 1 nM, about 20 pM toabout 950 pM, about 20 pM to about 900 pM, about 20 pM to about 850 pM,about 20 pM to about 800 pM, about 20 pM to about 750 pM, about 20 pM toabout 700 pM, about 20 pM to about 650 pM, about 20 pM to about 600 pM,about 20 pM to about 550 pM, about 20 pM to about 500 pM, about 20 pM toabout 450 pM, about 20 pM to about 400 pM, about 20 pM to about 350 pM,about 20 pM to about 300 pM, about 20 pM to about 250 pM, about 20 pM toabout 20 pM, about 200 pM to about 150 pM, about 20 pM to about 100 pM,about 20 pM to about 90 pM, about 20 pM to about 80 pM, about 20 pM toabout 70 pM, about 20 pM to about 60 pM, about 20 pM to about 50 pM,about 20 pM to about 40 pM, about 20 pM to about 30 pM, about 30 pM toabout 30 nM, about 30 pM to about 25 nM, about 30 pM to about 30 nM,about 30 pM to about 15 nM, about 30 pM to about 10 nM, about 30 pM toabout 5 nM, about 30 pM to about 2 nM, about 30 pM to about 1 nM, about30 pM to about 950 pM, about 30 pM to about 900 pM, about 30 pM to about850 pM, about 30 pM to about 800 pM, about 30 pM to about 750 pM, about30 pM to about 700 pM, about 30 pM to about 650 pM, about 30 pM to about600 pM, about 30 pM to about 550 pM, about 30 pM to about 500 pM, about30 pM to about 450 pM, about 30 pM to about 400 pM, about 30 pM to about350 pM, about 30 pM to about 300 pM, about 30 pM to about 250 pM, about30 pM to about 200 pM, about 30 pM to about 150 pM, about 30 pM to about100 pM, about 30 pM to about 90 pM, about 30 pM to about 80 pM, about 30pM to about 70 pM, about 30 pM to about 60 pM, about 30 pM to about 50pM, about 30 pM to about 40 pM, about 40 pM to about 30 nM, about 40 pMto about 25 nM, about 40 pM to about 30 nM, about 40 pM to about 15 nM,about 40 pM to about 10 nM, about 40 pM to about 5 nM, about 40 pM toabout 2 nM, about 40 pM to about 1 nM, about 40 pM to about 950 pM,about 40 pM to about 900 pM, about 40 pM to about 850 pM, about 40 pM toabout 800 pM, about 40 pM to about 750 pM, about 40 pM to about 700 pM,about 40 pM to about 650 pM, about 40 pM to about 600 pM, about 40 pM toabout 550 pM, about 40 pM to about 500 pM, about 40 pM to about 450 pM,about 40 pM to about 400 pM, about 40 pM to about 350 pM, about 40 pM toabout 300 pM, about 40 pM to about 250 pM, about 40 pM to about 200 pM,about 40 pM to about 150 pM, about 40 pM to about 100 pM, about 40 pM toabout 90 pM, about 40 pM to about 80 pM, about 40 pM to about 70 pM,about 40 pM to about 60 pM, about 40 pM to about 50 pM, about 50 pM toabout 30 nM, about 50 pM to about 25 nM, about 50 pM to about 30 nM,about 50 pM to about 15 nM, about 50 pM to about 10 nM, about 50 pM toabout 5 nM, about 50 pM to about 2 nM, about 50 pM to about 1 nM, about50 pM to about 950 pM, about 50 pM to about 900 pM, about 50 pM to about850 pM, about 50 pM to about 800 pM, about 50 pM to about 750 pM, about50 pM to about 700 pM, about 50 pM to about 650 pM, about 50 pM to about600 pM, about 50 pM to about 550 pM, about 50 pM to about 500 pM, about50 pM to about 450 pM, about 50 pM to about 400 pM, about 50 pM to about350 pM, about 50 pM to about 300 pM, about 50 pM to about 250 pM, about50 pM to about 200 pM, about 50 pM to about 150 pM, about 50 pM to about100 pM, about 50 pM to about 90 pM, about 50 pM to about 80 pM, about 50pM to about 70 pM, about 50 pM to about 60 pM, about 60 pM to about 30nM, about 60 pM to about 25 nM, about 60 pM to about 30 nM, about 60 pMto about 15 nM, about 60 pM to about 10 nM, about 60 pM to about 5 nM,about 60 pM to about 2 nM, about 60 pM to about 1 nM, about 60 pM toabout 950 pM, about 60 pM to about 900 pM, about 60 pM to about 850 pM,about 60 pM to about 800 pM, about 60 pM to about 750 pM, about 60 pM toabout 700 pM, about 60 pM to about 650 pM, about 60 pM to about 600 pM,about 60 pM to about 550 pM, about 60 pM to about 500 pM, about 60 pM toabout 450 pM, about 60 pM to about 400 pM, about 60 pM to about 350 pM,about 60 pM to about 300 pM, about 60 pM to about 250 pM, about 60 pM toabout 200 pM, about 60 pM to about 150 pM, about 60 pM to about 100 pM,about 60 pM to about 90 pM, about 60 pM to about 80 pM, about 60 pM toabout 70 pM, about 70 pM to about 30 nM, about 70 pM to about 25 nM,about 70 pM to about 30 nM, about 70 pM to about 15 nM, about 70 pM toabout 10 nM, about 70 pM to about 5 nM, about 70 pM to about 2 nM, about70 pM to about 1 nM, about 70 pM to about 950 pM, about 70 pM to about900 pM, about 70 pM to about 850 pM, about 70 pM to about 800 pM, about70 pM to about 750 pM, about 70 pM to about 700 pM, about 70 pM to about650 pM, about 70 pM to about 600 pM, about 70 pM to about 550 pM, about70 pM to about 500 pM, about 70 pM to about 450 pM, about 70 pM to about400 pM, about 70 pM to about 350 pM, about 70 pM to about 300 pM, about70 pM to about 250 pM, about 70 pM to about 200 pM, about 70 pM to about150 pM, about 70 pM to about 100 pM, about 70 pM to about 90 pM, about70 pM to about 80 pM, about 80 pM to about 30 nM, about 80 pM to about25 nM, about 80 pM to about 30 nM, about 80 pM to about 15 nM, about 80pM to about 10 nM, about 80 pM to about 5 nM, about 80 pM to about 2 nM,about 80 pM to about 1 nM, about 80 pM to about 950 pM, about 80 pM toabout 900 pM, about 80 pM to about 850 pM, about 80 pM to about 800 pM,about 80 pM to about 750 pM, about 80 pM to about 700 pM, about 80 pM toabout 650 pM, about 80 pM to about 600 pM, about 80 pM to about 550 pM,about 80 pM to about 500 pM, about 80 pM to about 450 pM, about 80 pM toabout 400 pM, about 80 pM to about 350 pM, about 80 pM to about 300 pM,about 80 pM to about 250 pM, about 80 pM to about 200 pM, about 80 pM toabout 150 pM, about 80 pM to about 100 pM, about 80 pM to about 90 pM,about 90 pM to about 30 nM, about 90 pM to about 25 nM, about 90 pM toabout 30 nM, about 90 pM to about 15 nM, about 90 pM to about 10 nM,about 90 pM to about 5 nM, about 90 pM to about 2 nM, about 90 pM toabout 1 nM, about 90 pM to about 950 pM, about 90 pM to about 900 pM,about 90 pM to about 850 pM, about 90 pM to about 800 pM, about 90 pM toabout 750 pM, about 90 pM to about 700 pM, about 90 pM to about 650 pM,about 90 pM to about 600 pM, about 90 pM to about 550 pM, about 90 pM toabout 500 pM, about 90 pM to about 450 pM, about 90 pM to about 400 pM,about 90 pM to about 350 pM, about 90 pM to about 300 pM, about 90 pM toabout 250 pM, about 90 pM to about 200 pM, about 90 pM to about 150 pM,about 90 pM to about 100 pM, about 100 pM to about 30 nM, about 100 pMto about 25 nM, about 100 pM to about 30 nM, about 100 pM to about 15nM, about 100 pM to about 10 nM, about 100 pM to about 5 nM, about 100pM to about 2 nM, about 100 pM to about 1 nM, about 100 pM to about 950pM, about 100 pM to about 900 pM, about 100 pM to about 850 pM, about100 pM to about 800 pM, about 100 pM to about 750 pM, about 100 pM toabout 700 pM, about 100 pM to about 650 pM, about 100 pM to about 600pM, about 100 pM to about 550 pM, about 100 pM to about 500 pM, about100 pM to about 450 pM, about 100 pM to about 400 pM, about 100 pM toabout 350 pM, about 100 pM to about 300 pM, about 100 pM to about 250pM, about 100 pM to about 200 pM, about 100 pM to about 150 pM, about150 pM to about 30 nM, about 150 pM to about 25 nM, about 150 pM toabout 30 nM, about 150 pM to about 15 nM, about 150 pM to about 10 nM,about 150 pM to about 5 nM, about 150 pM to about 2 nM, about 150 pM toabout 1 nM, about 150 pM to about 950 pM, about 150 pM to about 900 pM,about 150 pM to about 850 pM, about 150 pM to about 800 pM, about 150 pMto about 750 pM, about 150 pM to about 700 pM, about 150 pM to about 650pM, about 150 pM to about 600 pM, about 150 pM to about 550 pM, about150 pM to about 500 pM, about 150 pM to about 450 pM, about 150 pM toabout 400 pM, about 150 pM to about 350 pM, about 150 pM to about 300pM, about 150 pM to about 250 pM, about 150 pM to about 200 pM, about200 pM to about 30 nM, about 200 pM to about 25 nM, about 200 pM toabout 30 nM, about 200 pM to about 15 nM, about 200 pM to about 10 nM,about 200 pM to about 5 nM, about 200 pM to about 2 nM, about 200 pM toabout 1 nM, about 200 pM to about 950 pM, about 200 pM to about 900 pM,about 200 pM to about 850 pM, about 200 pM to about 800 pM, about 200 pMto about 750 pM, about 200 pM to about 700 pM, about 200 pM to about 650pM, about 200 pM to about 600 pM, about 200 pM to about 550 pM, about200 pM to about 500 pM, about 200 pM to about 450 pM, about 200 pM toabout 400 pM, about 200 pM to about 350 pM, about 200 pM to about 300pM, about 200 pM to about 250 pM, about 300 pM to about 30 nM, about 300pM to about 25 nM, about 300 pM to about 30 nM, about 300 pM to about 15nM, about 300 pM to about 10 nM, about 300 pM to about 5 nM, about 300pM to about 2 nM, about 300 pM to about 1 nM, about 300 pM to about 950pM, about 300 pM to about 900 pM, about 300 pM to about 850 pM, about300 pM to about 800 pM, about 300 pM to about 750 pM, about 300 pM toabout 700 pM, about 300 pM to about 650 pM, about 300 pM to about 600pM, about 300 pM to about 550 pM, about 300 pM to about 500 pM, about300 pM to about 450 pM, about 300 pM to about 400 pM, about 300 pM toabout 350 pM, about 400 pM to about 30 nM, about 400 pM to about 25 nM,about 400 pM to about 30 nM, about 400 pM to about 15 nM, about 400 pMto about 10 nM, about 400 pM to about 5 nM, about 400 pM to about 2 nM,about 400 pM to about 1 nM, about 400 pM to about 950 pM, about 400 pMto about 900 pM, about 400 pM to about 850 pM, about 400 pM to about 800pM, about 400 pM to about 750 pM, about 400 pM to about 700 pM, about400 pM to about 650 pM, about 400 pM to about 600 pM, about 400 pM toabout 550 pM, about 400 pM to about 500 pM, about 500 pM to about 30 nM,about 500 pM to about 25 nM, about 500 pM to about 30 nM, about 500 pMto about 15 nM, about 500 pM to about 10 nM, about 500 pM to about 5 nM,about 500 pM to about 2 nM, about 500 pM to about 1 nM, about 500 pM toabout 950 pM, about 500 pM to about 900 pM, about 500 pM to about 850pM, about 500 pM to about 800 pM, about 500 pM to about 750 pM, about500 pM to about 700 pM, about 500 pM to about 650 pM, about 500 pM toabout 600 pM, about 500 pM to about 550 pM, about 600 pM to about 30 nM,about 600 pM to about 25 nM, about 600 pM to about 30 nM, about 600 pMto about 15 nM, about 600 pM to about 10 nM, about 600 pM to about 5 nM,about 600 pM to about 2 nM, about 600 pM to about 1 nM, about 600 pM toabout 950 pM, about 600 pM to about 900 pM, about 600 pM to about 850pM, about 600 pM to about 800 pM, about 600 pM to about 750 pM, about600 pM to about 700 pM, about 600 pM to about 650 pM, about 700 pM toabout 30 nM, about 700 pM to about 25 nM, about 700 pM to about 30 nM,about 700 pM to about 15 nM, about 700 pM to about 10 nM, about 700 pMto about 5 nM, about 700 pM to about 2 nM, about 700 pM to about 1 nM,about 700 pM to about 950 pM, about 700 pM to about 900 pM, about 700 pMto about 850 pM, about 700 pM to about 800 pM, about 700 pM to about 750pM, about 800 pM to about 30 nM, about 800 pM to about 25 nM, about 800pM to about 30 nM, about 800 pM to about 15 nM, about 800 pM to about 10nM, about 800 pM to about 5 nM, about 800 pM to about 2 nM, about 800 pMto about 1 nM, about 800 pM to about 950 pM, about 800 pM to about 900pM, about 800 pM to about 850 pM, about 900 pM to about 30 nM, about 900pM to about 25 nM, about 900 pM to about 30 nM, about 900 pM to about 15nM, about 900 pM to about 10 nM, about 900 pM to about 5 nM, about 900pM to about 2 nM, about 900 pM to about 1 nM, about 900 pM to about 950pM, about 1 nM to about 30 nM, about 1 nM to about 25 nM, about 1 nM toabout 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about1 nM to about 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25nM, about 2 nM to about 20 nM, about 2 nM to about 15 nM, about 2 nM toabout 10 nM, about 2 nM to about 5 nM, about 4 nM to about 30 nM, about4 nM to about 25 nM, about 4 nM to about 20 nM, about 4 nM to about 15nM, about 4 nM to about 10 nM, about 4 nM to about 5 nM, about 5 nM toabout 30 nM, about 5 nM to about 25 nM, about 5 nM to about 20 nM, about5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 30nM, about 10 nM to about 25 nM, about 10 nM to about 20 nM, about 10 nMto about 15 nM, about 15 nM to about 30 nM, about 15 nM to about 25 nM,about 15 nM to about 20 nM, about 20 nM to about 30 nM, and about 20 nMto about 25 nM).

Any of the target-binding domains described herein can bind to itstarget with a K_(D) of between about 1 nM to about 10 nM (e.g., about 1nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM,about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM toabout 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM,about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM toabout 7 nM, about 3 nM to about 6 nM, about 3 nM to about 5 nM, about 3nM to about 4 nM, about 4 nM to about 10 nM, about 4 nM to about 9 nM,about 4 nM to about 8 nM, about 4 nM to about 7 nM, about 4 nM to about6 nM, about 4 nM to about 5 nM, about 5 nM to about 10 nM, about 5 nM toabout 9 nM, about 5 nM to about 8 nM, about 5 nM to about 7 nM, about 5nM to about 6 nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM,about 6 nM to about 8 nM, about 6 nM to about 7 nM, about 7 nM to about10 nM, about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM toabout 10 nM, about 8 nM to about 9 nM, and about 9 nM to about 10 nM).

A variety of different methods known in the art can be used to determinethe K_(D) values of any of the antigen-binding protein constructsdescribed herein (e.g., an electrophoretic mobility shift assay, afilter binding assay, surface plasmon resonance, and a biomolecularbinding kinetics assay, etc.).

Antigen-Binding Domains

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtarget-binding domain bind specifically to the same antigen. In someembodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the second target-binding domain bindspecifically to the same epitope. In some embodiments of thesemulti-chain chimeric polypeptides, the first target-binding domain andthe second target-binding domain include the same amino acid sequence.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtarget-binding domain bind specifically to different antigens.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, one or both of the first target-binding domain and thesecond target-binding domain is an antigen-binding domain. In someembodiments of any of the multi-chain chimeric polypeptides describedherein, the first target-binding domain and the second target-bindingdomain are each antigen-binding domains.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the antigen-binding domain includes or is a scFv or asingle domain antibody (e.g., a VHH or a VNAR domain).

In some examples, an antigen-binding domain (e.g., any of theantigen-binding domains described herein) can bind specifically to anyone of CD16a (see, e.g., those described in U.S. Pat. No. 9,035,026),CD28 (see, e.g., those described in U.S. Pat. No. 7,723,482), CD3 (see,e.g., those described in U.S. Pat. No. 9,226,962), CD33 (see, e.g.,those described in U.S. Pat. No. 8,759,494), CD20 (see, e.g., thosedescribed in WO 2014/026054), CD19 (see, e.g., those described in U.S.Pat. No. 9,701,758), CD22 (see, e.g., those described in WO2003/104425), CD123 (see, e.g., those described in WO 2014/130635),IL-1R (see, e.g., those described in U.S. Pat. No. 8,741,604), IL-1(see, e.g., those described in WO 2014/095808), VEGF (see, e.g., thosedescribed in U.S. Pat. No. 9,090,684), IL-6R (see, e.g., those describedin U.S. Pat. No. 7,482,436), IL-4 (see, e.g., those described in U.S.Patent Application Publication No. 2012/0171197), IL-10 (see, e.g.,those described in U.S. Patent Application Publication No.2016/0340413), PDL-1 (see, e.g., those described in Drees et al.,Protein Express. Purif. 94:60-66, 2014), TIGIT (see, e.g., thosedescribed in U.S. Patent Application Publication No. 2017/0198042), PD-1(see, e.g., those described in U.S. Pat. No. 7,488,802), TIM3 (see,e.g., those described in U.S. Pat. No. 8,552,156), CTLA4 (see, e.g.,those described in WO 2012/120125), MICA (see, e.g., those described inWO 2016/154585), MICB (see, e.g., those described in U.S. Pat. No.8,753,640), IL-6 (see, e.g., those described in Gejima et al., HumanAntibodies 11(4):121-129, 2002), IL-8 (see, e.g., those described inU.S. Pat. No. 6,117,980), TNFα (see, e.g., those described in Geng etal., Immunol. Res. 62(3):377-385, 2015), CD26a (see, e.g., thosedescribed in WO 2017/189526), CD36 (see, e.g., those described in U.S.Patent Application Publication No. 2015/0259429), ULBP2 (see, e.g.,those described in U.S. Pat. No. 9,273,136), CD30 (see, e.g., thosedescribed in Homach et al., Scand. J. Immunol. 48(5):497-501, 1998),CD200 (see, e.g., those described in U.S. Pat. No. 9,085,623), IGF-1R(see, e.g., those described in U.S. Patent Application Publication No.2017/0051063), MUC4AC (see, e.g., those described in WO 2012/170470),MUC5AC (see, e.g., those described in U.S. Pat. No. 9,238,084), Trop-2(see, e.g., those described in WO 2013/068946), CMET (see, e.g., thosedescribed in Edwardraja et al., Biotechnol. Bioeng. 106(3):367-375,2010), EGFR (see, e.g., those described in Akbari et al., Protein Expr.Purif. 127:8-15, 2016), HER1 (see, e.g., those described in U.S. PatentApplication Publication No. 2013/0274446), HER2 (see, e.g., thosedescribed in Cao et al., Biotechnol. Lett. 37(7):1347-1354, 2015), HER3(see, e.g., those described in U.S. Pat. No. 9,505,843), PSMA (see,e.g., those described in Parker et al., Protein Expr. Purif.89(2):136-145, 2013), CEA (see, e.g., those described in WO1995/015341), B7H3 (see, e.g., those described in U.S. Pat. No.9,371,395), EPCAM (see, e.g., those described in WO 2014/159531), BCMA(see, e.g., those described in Smith et al., Mol. Ther. 26(6):1447-1456,2018), P-cadherin (see, e.g., those described in U.S. Pat. No.7,452,537), CEACAM5 (see, e.g., those described in U.S. Pat. No.9,617,345), a UL16-binding protein (see, e.g., those described in WO2017/083612), HLA-DR (see, e.g., Pistillo et al., Exp. Clin.Immunogenet. 14(2):123-130, 1997), DLL4 (see, e.g., those described inWO 2014/007513), TYRO3 (see, e.g., those described in WO 2016/166348),AXL (see, e.g., those described in WO 2012/175692), MER (see, e.g.,those described in WO 2016/106221), CD122 (see, e.g., those described inU.S. Patent Application Publication No. 2016/0367664), CD155 (see, e.g.,those described in WO 2017/149538), or PDGF-DD (see, e.g., thosedescribed in U.S. Pat. No. 9,441,034).

The antigen-binding domains present in any of the multi-chain chimericpolypeptides described herein are each independently selected from thegroup consisting of: a VHH domain, a VNAR domain, and a scFv. In someembodiments, any of the antigen-binding domains described herein is aBiTe, a (scFv)₂, a nanobody, a nanobody-HSA, a DART, a TandAb, ascDiabody, a scDiabody-CH3, scFv-CH-CL-scFv, a HSAbody, scDiabody-HAS,or a tandem-scFv. Additional examples of antigen-binding domains thatcan be used in any of the multi-chain chimeric polypeptide are known inthe art.

A VHH domain is a single monomeric variable antibody domain that can befound in camelids. A VNAR domain is a single monomeric variable antibodydomain that can be found in cartilaginous fish. Non-limiting aspects ofVHH domains and VNAR domains are described in, e.g., Cromie et al.,Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp.Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol.32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015;Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah etal., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al.,Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol.74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci. 26:230-235,2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Rahbarizadeh etal., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al.,EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig.Drugs 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol. 911:15-26,2012; and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

In some embodiments, each of the antigen-binding domains in themulti-chain chimeric polypeptides described herein are both VHH domains,or at least one antigen-binding domain is a VHH domain. In someembodiments, each of the antigen-binding domains in the multi-chainchimeric polypeptides described herein are both VNAR domains, or atleast one antigen-binding domain is a VNAR domain. In some embodiments,each of the antigen-binding domains in the multi-chain chimericpolypeptides described herein are both scFv domains, or at least oneantigen-binding domain is a scFv domain.

In some embodiments, two or more of polypeptides present in themulti-chain chimeric polypeptide can assemble (e.g., non-covalentlyassemble) to form any of the antigen-binding domains described herein,e.g., an antigen-binding fragment of an antibody (e.g., any of theantigen-binding fragments of an antibody described herein), a VHH-scAb,a VHH-Fab, a Dual scFab, a F(ab′)2, a diabody, a crossMab, a DAF(two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a knobs-in-holescommon light chain, a knobs-in-holes assembly, a charge pair, a Fab-armexchange, a SEEDbody, a LUZ-Y, a Fcab, a κλ-body, an orthogonal Fab, aDVD-IgG, a IgG(H)-scFv, a scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG,IgG(L,H)-Fv, IgG(H)-V, V(H)—IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab,2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, a triplebody, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH,Fab-scFv, a F(ab′)2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalentHCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, an Intrabody, adock and lock, a 1 mmTAC, an IgG-IgG conjugate, a Cov-X-Body, and ascFv1-PEG-scFv2. See, e.g., Spiess et al., Mol. Immunol. 67:95-106,2015, incorporated in its entirety herewith, for a description of theseelements. Non-limiting examples of an antigen-binding fragment of anantibody include an Fv fragment, a Fab fragment, a F(ab′)2 fragment, anda Fab′ fragment. Additional examples of an antigen-binding fragment ofan antibody is an antigen-binding fragment of an IgG (e.g., anantigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., anantigen-binding fragment of a human or humanized IgG; e.g., human orhumanized IgG1, IgG2, IgG3, or IgG4); an antigen-binding fragment of anIgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., anantigen-binding fragment of a human or humanized IgA, e.g., a human orhumanized IgA1 or IgA2); an antigen-binding fragment of an IgD (e.g., anantigen-binding fragment of a human or humanized IgD); anantigen-binding fragment of an IgE (e.g., an antigen-binding fragment ofa human or humanized IgE); or an antigen-binding fragment of an IgM(e.g., an antigen-binding fragment of a human or humanized IgM).

An “Fv” fragment includes a non-covalently-linked dimer of one heavychain variable domain and one light chain variable domain.

A “Fab” fragment includes, the constant domain of the light chain andthe first constant domain (C_(H1)) of the heavy chain, in addition tothe heavy and light chain variable domains of the Fv fragment.

A “F(ab′)₂” fragment includes two Fab fragments joined, near the hingeregion, by disulfide bonds.

A “dual variable domain immunoglobulin” or “DVD-Ig” refers tomultivalent and multispecific binding proteins as described, e.g., inDiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al.,MABs 5:358-363, 2013; and U.S. Pat. Nos. 7,612,181; 8,258,268;8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each of whichis incorporated by reference in its entirety.

DARTs are described in, e.g., Garber, Nature Reviews Drug Discovery13:799-801, 2014.

In some embodiments of any of the antigen-binding domains describedherein can bind to an antigen selected from the group consisting of: aprotein, a carbohydrate, a lipid, and a combination thereof.

Additional examples and aspects of antigen-binding domains are known inthe art.

Soluble Interleukin or Cytokine Protein

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, one or both of the first target-binding domain and thesecond target-binding domain can be a soluble interleukin protein orsoluble cytokine protein. In some embodiments, the soluble interleukinor soluble cytokine protein is selected from the group of: IL-2, IL-3,IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, andFLT3L. Non-limiting examples of soluble IL-2, IL-3, IL-7, IL-8, IL-10,IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L are provided below.

Human Soluble IL-2 (SEQ ID NO: 9) aptssstkkt qlqlehllld lqmilnginnyknpkltrml tfkfympkka telkhlqcle eelkpleevl nlaqsknfhl rprdlisninvivlelkgse ttfmceyade tativeflnr witfcqsiis tlt Human Soluble IL-3 (SEQID NO: 10) apmtqttplkt swvncsnmid eiithlkqpp lplldfnnln gedqdilmennlrrpnleaf nravkslqna saiesilknl lpclplataa ptrhpihikd gdwnefrrkltfylktlena qaqqttlsla if Human Soluble IL-7 (SEQ ID NO: 11)dcdiegkdgkqyesv lmvsidqlld smkeigsncl nnefnffkrh icdankegmf lfraarklrqflkmnstgdf dlhllkvseg ttillnctgq vkgrkpaalg eaqptkslee nkslkeqkklndlcflkrll qeiktcwnki lmgtkeh Human Soluble IL-8 (SEQ ID NO: 12)egavlprsak elrcqcikty skpfhpkfik elrviesgph canteiivkl sdgrelcldpkenwvqrvve kflkraens Human Soluble IL-10 (SEQ ID NO: 13) spgqgtqsenscthfpgnlpnm lrdlrdafsr vktffqmkdq ldnlllkesl ledfkgylgc qalsemiqfyleevmpqaen qdpdikahvn slgenlktlr lrlrrchrfl pcenkskave qvknafnklqekgiykamse fdifinyiea ymtmkirn Human Soluble IL-15 (SEQ ID NO: 14)Nwvnvisdlkki edliqsmhid atlytesdvh psckvtamkc fllelqvisl esgdasihdtvenliilann slssngnvte sgckeceele eknikeflqs fvhivqmfin ts Human SolubleIL-17 (SEQ ID NO: 15) gitiprn pgcpnsedkn fprtvmvnln ihnrntntnpkrssdyynrs tspwnlhrne dperypsviw eakcrhlgci nadgnvdyhm nsvpiqqeilvlrrepphcp nsfrlekilv svgctcvtpi vhhva Human Soluble IL-18 (SEQ ID NO:16) yfgklesklsvirn lndqvlfidq gnrplfedmt dsdcrdnapr tifiismykdsqprgmavti svkcekistl scenkiisfk emnppdnikd tksdiiffqr svpghdnkmqfesssyegyf lacekerdlf klilkkedel gdrsimftvq ned Human Soluble PDGF-DD(SEQ ID NO: 17) rdtsatpqsasi kalrnanlrr desnhltdly rrdetiqvkg ngyvqsprfpnsyprnlllt wrlhsqentr iqlvfdnqfg leeaendicr ydfvevedis etstiirgrwcghkevppri ksrtnqikit fksddyfvak pgfkiyysll edfqpaaase tnwesvtssisgvsynspsv tdptliadal dkkiaefdtv edllkyfnpe swqedlenmy ldtpryrgrsyhdrkskvdl drlnddakry sctprnysvn ireelklanv vffprcllvq rcggncgcgtvnwrsctcns gktvkkyhev lqfepghikr rgraktmalv diqldhherc dcicssrppr HumanSoluble SCF (SEQ ID NO: 18) egicrnrvtnnvkdv tklvanlpkd ymitlkyvpgmdvlpshcwi semvvqlsds ltdlldkfsn iseglsnysi idklvnivdd lvecvkensskdlkksfksp eprlftpeef frifnrsida fkdfvvaset sdcvvsstls pekdsrvsvtkpfmlppvaa sslrndssss nrkaknppgd sslhwaamal palfsliigf afgalywkkrqpsltraven iqineednei smlqekeref qev Human Soluble FLT3L (SEQ ID NO: 19)tqdcsfqhspissd favkirelsd yllqdypvtv asnlqdeelc gglwrlvlaq rwmerlktvagskmqgller vnteihfvtk cafqpppscl rfvqtnisrl lqetseqlva lkpwitrqnfsrclelqcqp dsstlpppws prpleatapt apqpplllll llpvglllla aawclhwqrtrrrtprpgeq vppvpspqdl llveh

Non-limiting examples of soluble MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4,ULBP5, and ULBP6 are provided below.

Human Soluble MICA (SEQ ID NO: 20) ephslry nltvlswdgs vqsgfltevhldgqpflrcd rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkeglhslqeirvce ihednstrss qhfyydgelf lsqnletkew tmpqssraqt lamnvrnflkedamktkthy hamhadclqe lrrylksgvv lrrtvppmvn vtrseasegn itvtcrasgfypwnitlswr qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnhsthpvpsgkv lvlqshwqtf hvsavaaaai fviiifyvrc ckkktsaaeg pelvslqvldqhpvgtsdhr datqlgfqpl msdlgstgst ega Human Soluble MICB (SEQ ID NO: 21)aephslry nlmvlsqdes vqsgflaegh ldgqpflryd rqkrrakpqg qwaedvlgaktwdtetedlt engqdlrrtl thikdqkggl hslqeirvce ihedsstrgs rhfyydgelflsqnletqes tvpqssraqt lamnvtnfwk edamktkthy ramqadclqk lqrylksgvairrtvppmvn vtcsevsegn itvtcrassf yprnitltwr qdgvslshnt qqwgdvlpdgngtyqtwvat rirqgeeqrf tcymehsgnh gthpvpsgkv lvlqsqrtdf pyvsaampcfviiiilcvpc ckkktsaaeg pelvslqvld qhpvgtgdhr daaqlgfqpl msatgstgst egaHuman Soluble ULBP1 (SEQ ID NO: 22) wvdthclcydfiit pksrpepqwc evqglvderpflhydcvnhk akafaslgkk vnvtktweeq tetlrdvvdf lkgqlldiqv enlipiepltlqarmscehe ahghgrgswq flfngqkfll fdsnnrkwta lhpgakkmte kweknrdvtmffqkislgdc kmwleeflmy weqmldptkp pslapg Human Soluble ULBP2 (SEQ ID NO:23) gradphslcyditvi pkfrpgprwc avqgqvdekt flhydcgnkt vtpvsplgkklnvttawkaq npvlrevvdi lteqlrdiql enytpkeplt lqarmsceqk aeghssgswqfsfdgqifll fdsekrmwtt vhpgarkmke kwendkvvam sfhyfsmgdc igwledflmgmdstlepsag aplams Human Soluble ULBP3 (SEQ ID NO: 24) dahslwynftiihlprhgqqw cevqsqvdqk nflsydcgsd kvlsmghlee qlyatdawgk qlemlrevgqrlrleladte ledftpsgpl tlqvrmscec eadgyirgsw qfsfdgrkfl lfdsnnrkwtvvhagarrmk ekwekdsglt tffkmvsmrd ckswlrdflm hrkkrlepta pptmapg HumanSoluble ULBP4 (SEQ ID NO: 25) hslcfnftik slsrpgqpwc eaqvflnknlflqynsdnnm vkplgllgkk vyatstwgel tqtlgevgrd lrmllcdikp qiktsdpstlqvemfcqrea erctgaswqf atngeksllf damnmtwtvi nheaskiket wkkdrglekyfrklskgdcd hwlreflghw eampeptvsp vnasdihwss sslpdrwiil gafillvlmgivlicvwwqn gewqaglwpl rts Human Soluble ULBP5 (SEQ ID NO: 26) gladphslcyditvi pkfrpgprwc avqgqvdekt flhydcgskt vtpvsplgkk lnvttawkaqnpvlrevvdi lteqlldiql enyipkeplt lqarmsceqk aeghgsgswq lsfdgqifllfdsenrmwtt vhpgarkmke kwendkdmtm sfhyismgdc tgwledflmg mdstlepsagapptmssg Human Soluble ULBP6 (SEQ ID NO: 27) rrddp hslcyditvi pkfrpgprwcavqgqvdekt flhydcgnkt vtpvsplgkk lnvtmawkaq npvlrevvdi lteqlldiqlenytpkeplt lqarmsceqk aeghssgswq fsidgqtfll fdsekrmwtt vhpgarkmkekwendkdvam sfhyismgdc igwledflmg mdstlepsag aplamssg

Additional examples of soluble interleukin proteins and soluble cytokineproteins are known in the art.

Soluble Receptor

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, one or both of the first target-binding domain and thesecond target-binding domain is a soluble interleukin receptor, asoluble cytokine receptor or a ligand receptor. In some embodiments, thesoluble receptor is a soluble TGF-β receptor II (TGF-β RII) (see, e.g.,those described in Yung et al., Am. J. Resp. Crit. Care Med.194(9):1140-1151, 2016), a soluble TGF-βRIII (see, e.g., those describedin Heng et al., Placenta 57:320, 2017), a soluble NKG2D (see, e.g.,Cosman et al., Immunity 14(2):123-133, 2001; Costa et al., Front.Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389/fimmu.2018.01150), a soluble NKp30 (see, e.g., Costa et al.,Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389/fimmu.2018.01150), a soluble NKp44 (see, e.g., those describedin Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018;doi: 10.3389/fimmu.2018.01150), a soluble NKp46 (see, e.g., Mandelboimet al., Nature 409:1055-1060, 2001; Costa et al., Front. Immunol., Vol.9, Article 1150, May 29, 2018; doi: 10.3389/fimmu.2018.01150), a solubleDNAM-1 (see, e.g., those described in Costa et al., Front. Immunol.,Vol. 9, Article 1150, May 29, 2018; doi: 10.3389/fimmu.2018.01150), ascMHCI (see, e.g., those described in Washburn et al., PLoS One6(3):e18439, 2011), a scMHCII (see, e.g., those described in Bishwajitet al., Cellular Immunol. 170(1):25-33, 1996), a scTCR (see, e.g., thosedescribed in Weber et al., Nature 356(6372):793-796, 1992), a solubleCD155 (see, e.g., those described in Tahara-Hanaoka et al., Int.Immunol. 16(4):533-538, 2004), or a soluble CD28 (see, e.g., Hebbar etal., Clin. Exp. Immunol. 136:388-392, 2004).

Additional examples of soluble interleukin receptors and solublecytokine receptors are known in the art.

Additional Antigen-Binding Domains

In some embodiments of any of the multi-chain chimeric polypeptides, thefirst chimeric polypeptide further includes one or more (e.g., two,three, four, five, six, seven, eight, nine, or ten) additionaltarget-binding domain(s) (e.g., any of the exemplary target-bindingdomains described herein or known in the art), where at least one of theone or more additional antigen-binding domain(s) is positioned betweenthe soluble tissue factor domain (e.g., any of the exemplary solubletissue factor domains described herein or known in the art) and thefirst domain of the pair of affinity domains (e.g., any of the exemplaryfirst domains of any of the exemplary pairs of affinity domainsdescribed herein). In some embodiments, the first chimeric polypeptidecan further include a linker sequence (e.g., any of the exemplary linkersequences described herein or known in the art) between the solubletissue factor domain (e.g., any of the exemplary soluble tissue factordomains described herein) and the at least one of the one or moreadditional target-binding domain(s) (e.g., any of the exemplarytarget-binding domains described herein or known in the art), and/or alinker sequence (e.g., any of the exemplary linker sequences describedherein or known in the art) between the at least one of the one or moreadditional target-binding domain(s) (e.g., any of the exemplarytarget-binding domains described herein or known in the art) and thefirst domain of the pair of affinity domains (e.g., any of the exemplaryfirst domains described herein of any of the exemplary pairs of affinitydomains described herein).

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first chimeric polypeptide further includes one ormore (e.g., two, three, four, five, six, seven, eight, nine, or ten)additional target-binding domains at the N-terminal and/or C-terminalend of the first chimeric polypeptide. In some embodiments, at least oneof the one or more additional target-binding domains (e.g., any of theexemplary target-binding domains described herein or known in the art)directly abuts the first domain of the pair of affinity domains (e.g.,any of the exemplary first domains described herein of any of theexemplary pairs of affinity domains described herein) in the firstchimeric polypeptide. In some embodiments, the first chimericpolypeptide further includes a linker sequence (e.g., any of theexemplary linker sequences described herein or known in the art) betweenthe at least one of the one or more additional target-binding domains(e.g., any of the exemplary target-binding domains described herein orknown in the art) and the first domain of the pair of affinity domains(e.g., any of the exemplary first domains described herein of any of theexemplary pairs of affinity domains described herein). In someembodiments, the at least one of the one or more additionaltarget-binding domains (e.g., any of the exemplary target-bindingdomains described herein or known in the art) directly abuts the firsttarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art) in the first chimeric polypeptide.In some embodiments, the first chimeric polypeptide further comprises alinker sequence (e.g., any of the exemplary linker sequences describedherein or known in the art) between the at least one of the one or moreadditional target-binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) and thefirst target-binding domain (e.g., any of the exemplary target-bindingdomains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, at least one of the one or more additionaltarget-binding domains (e.g., any of the exemplary target-bindingdomains described herein or known in the art) is disposed at the N-and/or C-terminus of the first chimeric polypeptide, and at least one ofthe one or more additional target-binding domains (e.g., any of theexemplary target-binding domains described herein or known in the art)is positioned between the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains described herein or known in theart) and the first domain of the pair of affinity domains (e.g., any ofthe exemplary first domains of any of the exemplary pairs of affinitydomains described herein) in the first chimeric polypeptide. In someembodiments, the at least one additional target-binding domain (e.g.,any of the exemplary target-binding domains described herein or known inthe art) of the one or more additional target-binding domains disposedat the N-terminus directly abuts the first target-binding domain (e.g.,any of the exemplary target-binding domains described herein or known inthe art) or the first domain of the pair of affinity domains (e.g., anyof the exemplary first domains described herein of any of the exemplarypairs of affinity domains described herein) in the first chimericpolypeptide. In some embodiments, the first chimeric polypeptide furthercomprises a linker sequence (e.g., any of the linker sequences describedherein or known in the art) disposed between the at least one additionaltarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art) and the first target-bindingdomain (e.g., any of the exemplary target-binding domains describedherein or known in the art) or the first domain of the pair of affinitydomains (e.g., any of the exemplary first domains described herein ofany of the exemplary pairs of affinity domains described herein) in thefirst chimeric polypeptide. In some embodiments, the at least oneadditional target-binding domain (e.g., any of the exemplarytarget-binding domains described herein or known in the art) of the oneor more additional target-binding domains disposed at the C-terminusdirectly abuts the first target-binding domain (e.g., any of theexemplary target-binding domains described herein or known in the art)or the first domain of the pair of affinity domains (e.g., any of theexemplary first domains of any of the exemplary pairs of affinitydomains described herein) in the first chimeric polypeptide. In someembodiments, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linker sequences described hereinor known in the art) disposed between the at least one additionaltarget-binding domain (e.g., any of the exemplary target-binding domainsdescribed herein or known in the art) and the first target-bindingdomain (e.g., any of the exemplary target-binding domains describedherein or known in the art) or the first domain of the pair of affinitydomains (e.g., any of the exemplary first domains described herein ofany of the exemplary pairs of affinity domains described herein) in thefirst chimeric polypeptide. In some embodiments, the at least one of theone or more additional target-binding domains (e.g., any of theexemplary target-binding domains described herein or known in the art)positioned between the soluble tissue factor domain (e.g., any of theexemplary soluble tissue factor domains described herein) and the firstdomain of the pair of affinity domains (e.g., any of the first domainsdescribed herein or any of the exemplary pairs of affinity domainsdescribed herein), directly abuts the soluble tissue factor domainand/or the first domain of the pair of affinity domains. In someembodiments, the first chimeric polypeptide further comprises a linkersequence (e.g., any of the exemplary linker sequences described hereinor known in the art) disposed (i) between the soluble tissue factordomain (e.g., any of the exemplary soluble tissue factor domainsdescribed herein) and the at least one of the one or more additionaltarget-binding domains (e.g., any of the exemplary target-bindingdomains described herein or known in the art) positioned between thesoluble tissue factor domain (e.g., any of the exemplary soluble tissuefactor domains described herein) and the first domain of the pair ofaffinity domains (e.g., any of the exemplary first domains of any of theexemplary pairs of affinity domains described herein), and/or (ii)between the first domain of the pair of affinity domains and the atleast one of the one or more additional target-binding domainspositioned between the soluble tissue factor domain and the first domainof the pair of affinity domains.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the second chimeric polypeptide further includes oneor more (e.g., two, three, four, five, six, seven, eight, nine, or ten)additional target-binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) at theN-terminal end and/or the C-terminal end of the second chimericpolypeptide. In some embodiments, at least one of the one or moreadditional target-binding domains (e.g., any of the exemplarytarget-binding domains described herein or known in the art) directlyabuts the second domain of the pair of affinity domains (e.g., any ofthe exemplary second domains of any of the exemplary pairs of affinitydomains described herein) in the second chimeric polypeptide. In someembodiments, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linker sequences described hereinor known in the art) between at least one of the one or more additionaltarget-binding domains (e.g., any of the exemplary target-bindingdomains described herein or known in the art) and the second domain ofthe pair of affinity domains (e.g., any of the second domains describedherein of any of the exemplary pairs of affinity domains describedherein) in the second chimeric polypeptide. In some embodiments, atleast one of the one or more additional target-binding domains (e.g.,any of the exemplary target-binding domains described herein or known inthe art) directly abuts the second target-binding domain (e.g., any ofthe target-binding domains described herein or known in the art) in thesecond chimeric polypeptide. In some embodiments, the second chimericpolypeptide further includes a linker sequence (e.g., any of theexemplary linker sequences described herein or known in the art) betweenat least one of the one or more additional target-binding domains (e.g.,any of the exemplary target binding domains described herein or known inthe art) and the second target-binding domain (e.g., any of theexemplary target binding domains described herein or known in the art)in the second chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, two or more (e.g., three or more, four or more, fiveor more, six or more, seven or more, eight or more, nine or more, or tenor more) of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same antigen. In some embodiments, two or more(e.g., three or more, four or more, five or more, six or more, seven ormore, eight or more, nine or more, or ten or more) of the firsttarget-binding domain, the second target-binding domain, and the one ormore additional target-binding domains bind specifically to the sameepitope. In some embodiments, two or more (e.g., three or more, four ormore, five or more, six or more, seven or more, eight or more, nine ormore, or ten or more) of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains include the same amino acid sequence. In some embodiments, thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains each bind specifically tothe same antigen. In some embodiments, the first target-binding domain,the second target-binding domain, and the one or more additionaltarget-binding domains each bind specifically to the same epitope. Insome embodiments, the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains each include the same amino acid sequence.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains bind specifically to different antigens. In some embodiments ofany of the multi-chain chimeric polypeptides described herein, one ormore (e.g., two or more, three or more, four or more, five or more, sixor more, seven or more, eight or more, nine or more, or ten or more) ofthe first target-binding domain, the second target-binding domain, andthe one or more target-binding domains is an antigen-binding domain. Insome embodiments, the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains are each an antigen-binding domain (e.g., a scFv or asingle-domain antibody).

Pairs of Affinity Domains

In some embodiments, a multi-chain chimeric polypeptide includes: 1) afirst chimeric polypeptide that includes a first domain of a pair ofaffinity domains, and 2) a second chimeric polypeptide that includes asecond domain of a pair of affinity domains such that the first chimericpolypeptide and the second chimeric polypeptide associate through thebinding of the first domain and the second domain of the pair ofaffinity domains. In some embodiments, the pair of affinity domains is asushi domain from an alpha chain of human IL-15 receptor (IL15Rα) and asoluble IL-15. A sushi domain, also known as a short consensus repeat ortype 1 glycoprotein motif, is a common motif in protein-proteininteraction. Sushi domains have been identified on a number ofprotein-binding molecules, including complement components C1r, C1s,factor H, and C2m, as well as the nonimmunologic molecules factor XIIIand β2-glycoprotein. A typical Sushi domain has approximately 60 aminoacid residues and contains four cysteines (Ranganathan, Pac. SympBiocomput. 2000:155-67). The first cysteine can form a disulfide bondwith the third cysteine, and the second cysteine can form a disulfidebridge with the fourth cysteine. In some embodiments in which one memberof the pair of affinity domains is a soluble IL-15, the soluble IL15 hasa D8N or D8A amino acid substitution. In some embodiments in which onemember of the pair of affinity domains is an alpha chain of human IL-15receptor (IL15Rα), the human IL15Rα is a mature full-length IL15Rα. Insome embodiments, the pair of affinity domains is barnase and barnstar.In some embodiments, the pair of affinity domains is a PKA and an AKAP.In some embodiments, the pair of affinity domains is an adapter/dockingtag module based on mutated RNase I fragments (Rossi, Proc Natl Acad SciUSA. 103:6841-6846, 2006; Sharkey et al., Cancer Res. 68:5282-5290,2008; Rossi et al., Trends Pharmacol Sci. 33:474-481, 2012) or SNAREmodules based on interactions of the proteins syntaxin, synaptotagmin,synaptobrevin, and SNAP25 (Deyev et al., Nat Biotechnol. 1486-1492,2003).

In some embodiments, a first chimeric polypeptide of a multi-chainchimeric polypeptide includes a first domain of a pair of affinitydomains and a second chimeric polypeptide of the multi-chain chimericpolypeptide includes a second domain of a pair of affinity domains,wherein the first domain of the pair of affinity domains and the seconddomain of the pair of affinity domains bind to each other with adissociation equilibrium constant (K_(D)) of less than 1×10⁻⁷ M, lessthan 1×10⁻⁸M, less than 1×10⁻⁹ M, less than 1×10⁻¹⁰ M, less than 1×10⁻¹¹M, less than 1×10⁻¹²M, or less than 1×10⁻¹³ M. In some embodiments, thefirst domain of the pair of affinity domains and the second domain ofthe pair of affinity domains bind to each other with a K_(D) of about1×10⁻⁴ M to about 1×10⁻⁶M, about 1×10⁻⁵M to about 1×10⁻⁷M, about 1×10⁻⁶M to about 1×10⁻⁸ M, about 1×10⁻⁷M to about 1×10⁻⁹ M, about 1×10⁻⁸M toabout 1×10⁻¹⁰ M, about 1×10⁻⁹M to about 1×10⁻¹¹ M, about 1×10⁻¹⁰ M toabout 1×10⁻¹² M, about 1×10⁻¹¹ M to about 1×10⁻¹³ M, about 1×10⁻⁴ M toabout 1×10⁻⁵ M, about 1×10⁻⁵ M to about 1×10⁻⁶ M, about 1×10⁻⁶ M toabout 1×10⁻⁷ M, about 1×10⁻⁷M to about 1×10⁻⁸M, about 1×10⁻⁸M to about1×10⁻⁹ M, about 1×10⁻⁹M to about 1×10⁻¹⁰ M, about 1×10⁻¹⁰ M to about1×10⁻¹¹M, about 1×10⁻¹¹M to about 1×10⁻¹²M, or about 1×10⁻¹²M to about1×10⁻¹³ M (inclusive). Any of a variety of different methods known inthe art can be used to determine the K_(D) value of the binding of thefirst domain of the pair of affinity domains and the second domain ofthe pair of affinity domains (e.g., an electrophoretic mobility shiftassay, a filter binding assay, surface plasmon resonance, and abiomolecular binding kinetics assay, etc.).

In some embodiments, a first chimeric polypeptide of a multi-chainchimeric polypeptide includes a first domain of a pair of affinitydomains and a second chimeric polypeptide of the multi-chain chimericpolypeptide includes a second domain of a pair of affinity domains,wherein the first domain of the pair of affinity domains, the seconddomain of the pair of affinity domains, or both is about 10 to 100 aminoacids in length. For example, a first domain of a pair of affinitydomains, a second domain of a pair of affinity domains, or both can beabout 10 to 100 amino acids in length, about 15 to 100 amino acids inlength, about 20 to 100 amino acids in length, about 25 to 100 aminoacids in length, about 30 to 100 amino acids in length, about 35 to 100amino acids in length, about 40 to 100 amino acids in length, about 45to 100 amino acids in length, about 50 to 100 amino acids in length,about 55 to 100 amino acids in length, about 60 to 100 amino acids inlength, about 65 to 100 amino acids in length, about 70 to 100 aminoacids in length, about 75 to 100 amino acids in length, about 80 to 100amino acids in length, about 85 to 100 amino acids in length, about 90to 100 amino acids in length, about 95 to 100 amino acids in length,about 10 to 95 amino acids in length, about 10 to 90 amino acids inlength, about 10 to 85 amino acids in length, about 10 to 80 amino acidsin length, about 10 to 75 amino acids in length, about 10 to 70 aminoacids in length, about 10 to 65 amino acids in length, about 10 to 60amino acids in length, about 10 to 55 amino acids in length, about 10 to50 amino acids in length, about 10 to 45 amino acids in length, about 10to 40 amino acids in length, about 10 to 35 amino acids in length, about10 to 30 amino acids in length, about 10 to 25 amino acids in length,about 10 to 20 amino acids in length, about 10 to 15 amino acids inlength, about 20 to 30 amino acids in length, about 30 to 40 amino acidsin length, about 40 to 50 amino acids in length, about 50 to 60 aminoacids in length, about 60 to 70 amino acids in length, about 70 to 80amino acids in length, about 80 to 90 amino acids in length, about 90 to100 amino acids in length, about 20 to 90 amino acids in length, about30 to 80 amino acids in length, about 40 to 70 amino acids in length,about 50 to 60 amino acids in length, or any range in between. In someembodiments, a first domain of a pair of affinity domains, a seconddomain of a pair of affinity domains, or both is about 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 aminoacids in length.

In some embodiments, any of the first and/or second domains of a pair ofaffinity domains disclosed herein can include one or more additionalamino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) atits N-terminus and/or C-terminus, so long as the function of the firstand/or second domains of a pair of affinity domains remains intact. Forexample, a sushi domain from an alpha chain of human IL-15 receptor(IL15Rα) can include one or more additional amino acids at theN-terminus and/or the C-terminus, while still retaining the ability tobind to a soluble IL-15. Additionally or alternatively, a soluble IL-15can include one or more additional amino acids at the N-terminus and/orthe C-terminus, while still retaining the ability to bind to a sushidomain from an alpha chain of human IL-15 receptor (IL15Rα).

A non-limiting example of a sushi domain from an alpha chain of IL-15receptor alpha (IL15Rα) can include a sequence that is at least 70%identical, at least 75% identical, at least 80% identical, at least 85%identical, at least 90% identical, at least 95% identical, at least 99%identical, or 100% identical toITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH WTTPSLKCIR (SEQID NO: 28). In some embodiments, a sushi domain from an alpha chain ofIL15Rα can be encoded by a nucleic acid including

(SEQ ID NO: 29) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG.

In some embodiments, a soluble IL-15 can include a sequence that is atleast 70% identical, at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, atleast 99% identical, or 100% identical toNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINT S (SEQ ID NO:14). In some embodiments, a soluble IL-15 can be encoded by a nucleicacid including the sequence of

(SEQ ID NO: 30) AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC.Signal Sequence

In some embodiments, a multi-chain chimeric polypeptide includes a firstchimeric polypeptide that includes a signal sequence at its N-terminalend. In some embodiments, a multi-chain chimeric polypeptide includes asecond chimeric polypeptide that includes a signal sequence at itsN-terminal end. In some embodiments, both the first chimeric polypeptideof a multi-chain chimeric polypeptide and a second chimeric polypeptideof the multi-chain chimeric polypeptide include a signal sequence. Aswill be understood by those of ordinary skill in the art, a signalsequence is an amino acid sequence that is present at the N-terminus ofa number of endogenously produced proteins that directs the protein tothe secretory pathway (e.g., the protein is directed to reside incertain intracellular organelles, to reside in the cell membrane, or tobe secreted from the cell). Signal sequences are heterogeneous anddiffer greatly in their primary amino acid sequences. However, signalsequences are typically 16 to 30 amino acids in length and include ahydrophilic, usually positively charged N-terminal region, a centralhydrophobic domain, and a C-terminal region that contains the cleavagesite for signal peptidase.

In some embodiments, a first chimeric polypeptide of a multi-chainchimeric polypeptide, a second chimeric polypeptide of the multi-chainchimeric polypeptide, or both includes a signal sequence having an aminoacid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 31). In some embodiments, afirst chimeric polypeptide of a multi-chain chimeric polypeptide, asecond chimeric polypeptide of the multi-chain chimeric polypeptide, orboth includes a signal sequence encoded by the nucleic acid sequence

(SEQ ID NO: 32) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTA CTCC,(SEQ ID NO: 33) ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTA CAGC,or (SEQ ID NO: 34) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC.

In some embodiments, a first chimeric polypeptide of a multi-chainchimeric polypeptide, a second chimeric polypeptide of the multi-chainchimeric polypeptide, or both includes a signal sequence having an aminoacid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 35). In some embodiments, afirst chimeric polypeptide of a multi-chain chimeric polypeptide, asecond chimeric polypeptide of the multi-chain chimeric polypeptide, orboth includes a signal sequence having an amino acid sequenceMGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (SEQ ID NO:36). In some embodiments, a first chimeric polypeptide of a multi-chainchimeric polypeptide, a second chimeric polypeptide of the multi-chainchimeric polypeptide, or both includes a signal sequence having an aminoacid sequence

(SEQ ID NO: 37) MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG.In some embodiments, a first chimeric polypeptide of a multi-chainchimeric polypeptide, a second chimeric polypeptide of the multi-chainchimeric polypeptide, or both includes a signal sequence having an aminoacid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 38). Those ofordinary skill in the art will be aware of other appropriate signalsequences for use in a first chimeric polypeptide and/or a secondchimeric polypeptide of multi-chain chimeric polypeptides describedherein.

In some embodiments, a first chimeric polypeptide of a multi-chainchimeric polypeptide, a second chimeric polypeptide of the multi-chainchimeric polypeptide, or both includes a signal sequence that is about10 to 100 amino acids in length. For example, a signal sequence can beabout 10 to 100 amino acids in length, about 15 to 100 amino acids inlength, about 20 to 100 amino acids in length, about 25 to 100 aminoacids in length, about 30 to 100 amino acids in length, about 35 to 100amino acids in length, about 40 to 100 amino acids in length, about 45to 100 amino acids in length, about 50 to 100 amino acids in length,about 55 to 100 amino acids in length, about 60 to 100 amino acids inlength, about 65 to 100 amino acids in length, about 70 to 100 aminoacids in length, about 75 to 100 amino acids in length, about 80 to 100amino acids in length, about 85 to 100 amino acids in length, about 90to 100 amino acids in length, about 95 to 100 amino acids in length,about 10 to 95 amino acids in length, about 10 to 90 amino acids inlength, about 10 to 85 amino acids in length, about 10 to 80 amino acidsin length, about 10 to 75 amino acids in length, about 10 to 70 aminoacids in length, about 10 to 65 amino acids in length, about 10 to 60amino acids in length, about 10 to 55 amino acids in length, about 10 to50 amino acids in length, about 10 to 45 amino acids in length, about 10to 40 amino acids in length, about 10 to 35 amino acids in length, about10 to 30 amino acids in length, about 10 to 25 amino acids in length,about 10 to 20 amino acids in length, about 10 to 15 amino acids inlength, about 20 to 30 amino acids in length, about 30 to 40 amino acidsin length, about 40 to 50 amino acids in length, about 50 to 60 aminoacids in length, about 60 to 70 amino acids in length, about 70 to 80amino acids in length, about 80 to 90 amino acids in length, about 90 to100 amino acids in length, about 20 to 90 amino acids in length, about30 to 80 amino acids in length, about 40 to 70 amino acids in length,about 50 to 60 amino acids in length, or any range in between. In someembodiments, a signal sequence is about 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

In some embodiments, any of the signal sequences disclosed herein caninclude one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8,9, 10, or more amino acids) at its N-terminus and/or C-terminus, so longas the function of the signal sequence remains intact. For example, asignal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ IDNO: 35) can include one or more additional amino acids at the N-terminusor C-terminus, while still retaining the ability to direct a firstchimeric polypeptide of a multi-chain chimeric polypeptide, a secondchimeric polypeptide of the multi-chain chimeric polypeptide, or both tothe secretory pathway.

In some embodiments, a first chimeric polypeptide of a multi-chainchimeric polypeptide, a second chimeric polypeptide of the multi-chainchimeric polypeptide, or both includes a signal sequence that directsthe multi-chain chimeric polypeptide into the extracellular space. Suchembodiments are useful in producing multi-chain chimeric polypeptidesthat are relatively easy to be isolated and/or purified.

Peptide Tags

In some embodiments, a multi-chain chimeric polypeptide includes a firstchimeric polypeptide that includes a peptide tag (e.g., at theN-terminal end or the C-terminal end of the first chimeric polypeptide).In some embodiments, a multi-chain chimeric polypeptide includes asecond chimeric polypeptide that includes a peptide tag (e.g., at theN-terminal end or the C-terminal end of the second chimericpolypeptide). In some embodiments, both the first chimeric polypeptideof a multi-chain chimeric polypeptide and a second chimeric polypeptideof the multi-chain chimeric polypeptide include a peptide tag. In someembodiments, a first chimeric polypeptide of a multi-chain chimericpolypeptide, a second chimeric polypeptide of the multi-chain chimericpolypeptide, or both include two or more peptide tags.

Exemplary peptide tags that can be included in a first chimericpolypeptide of a multi-chain chimeric polypeptide, a second chimericpolypeptide of the multi-chain chimeric polypeptide, or both include,without limitation, AviTag (GLNDIFEAQKIEWHE; SEQ ID NO: 39), acalmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 40), apolyglutamate tag (EEEEEE; SEQ ID NO: 41), an E-tag (GAPVPYPDPLEPR; SEQID NO: 42), a FLAG-tag (DYKDDDDK; SEQ ID NO: 43), an HA-tag, a peptidefrom hemagglutinin (YPYDVPDYA; SEQ ID NO: 44), a his-tag (HHHHH (SEQ IDNO: 45); HHHHHH (SEQ ID NO: 46); HHHHHHH (SEQ ID NO: 47); HHHHHHHH (SEQID NO: 48); HHHHHHHHH (SEQ ID NO: 49); or HHHHHHHHHH (SEQ ID NO: 50)), amyc-tag (EQKLISEEDL; SEQ ID NO: 51), NE-tag (TKENPRSNQEESYDDNES; SEQ IDNO: 52), S-tag, (KETAAAKFERQHMDS; SEQ ID NO: 53), SBP-tag(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO: 54), Softag 1(SLAELLNAGLGGS; SEQ ID NO: 55), Softag 3 (TQDPSRVG; SEQ ID NO: 56),Spot-tag (PDRVRAVSHWSS; SEQ ID NO: 57), Strep-tag (WSHPQFEK; SEQ ID NO:58), TC tag (CCPGCC; SEQ ID NO: 59), Ty tag (EVHTNQDPLD; SEQ ID NO: 60),V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 61), VSV-tag (YTDIEMNRLGK; SEQ ID NO:62), and Xpress tag (DLYDDDDK; SEQ ID NO: 63). In some embodiments,tissue factor protein is a peptide tag.

Peptide tags that can be included in a first chimeric polypeptide of amulti-chain chimeric polypeptide, a second chimeric polypeptide of themulti-chain chimeric polypeptide, or both can be used in any of avariety of applications related to the multi-chain chimeric polypeptide.For example, a peptide tag can be used in the purification of amulti-chain chimeric polypeptide. As one non-limiting example, a firstchimeric polypeptide of a multi-chain chimeric polypeptide (e.g., arecombinantly expressed first chimeric polypeptide), a second chimericpolypeptide of the multi-chain chimeric polypeptide (e.g., arecombinantly expressed second chimeric polypeptide), or both caninclude a myc tag; the multi-chain chimeric polypeptide that includesthe myc-tagged first chimeric polypeptide, the myc-tagged secondchimeric polypeptide, or both can be purified using an antibody thatrecognizes the myc tag(s). One non-limiting example of an antibody thatrecognizes a myc tag is 9E10, available from the non-commercialDevelopmental Studies Hybridoma Bank. As another non-limiting example, afirst chimeric polypeptide of a multi-chain chimeric polypeptide (e.g.,a recombinantly expressed first chimeric polypeptide), a second chimericpolypeptide of the multi-chain chimeric polypeptide (e.g., arecombinantly expressed second chimeric polypeptide), or both caninclude a histidine tag; the multi-chain chimeric polypeptide thatincludes the histidine-tagged first chimeric polypeptide, thehistidine-tagged second chimeric polypeptide, or both can be purifiedusing a nickel or cobalt chelate. Those of ordinary skill in the artwill be aware of other suitable tags and agent that bind those tags foruse in purifying multi-chain chimeric polypeptide. In some embodiments,a peptide tag is removed from the first chimeric polypeptide and/or thesecond chimeric polypeptide of the multi-chain chimeric polypeptideafter purification. In some embodiments, a peptide tag is not removedfrom the first chimeric polypeptide and/or the second chimericpolypeptide of the multi-chain chimeric polypeptide after purification.

Peptide tags that can be included in a first chimeric polypeptide of amulti-chain chimeric polypeptide, a second chimeric polypeptide of themulti-chain chimeric polypeptide, or both can be used, for example, inimmunoprecipitation of the multi-chain chimeric polypeptide, imaging ofthe multi-chain chimeric polypeptide (e.g., via Western blotting, ELISA,flow cytometry, and/or immunocytochemistry), and/or solubilization ofthe multi-chain chimeric polypeptide.

In some embodiments, a first chimeric polypeptide of a multi-chainchimeric polypeptide, a second chimeric polypeptide of the multi-chainchimeric polypeptide, or both includes a peptide tag that is about 10 to100 amino acids in length. For example, a peptide tag can be about 10 to100 amino acids in length, about 15 to 100 amino acids in length, about20 to 100 amino acids in length, about 25 to 100 amino acids in length,about 30 to 100 amino acids in length, about 35 to 100 amino acids inlength, about 40 to 100 amino acids in length, about 45 to 100 aminoacids in length, about 50 to 100 amino acids in length, about 55 to 100amino acids in length, about 60 to 100 amino acids in length, about 65to 100 amino acids in length, about 70 to 100 amino acids in length,about 75 to 100 amino acids in length, about 80 to 100 amino acids inlength, about 85 to 100 amino acids in length, about 90 to 100 aminoacids in length, about 95 to 100 amino acids in length, about 10 to 95amino acids in length, about 10 to 90 amino acids in length, about 10 to85 amino acids in length, about 10 to 80 amino acids in length, about 10to 75 amino acids in length, about 10 to 70 amino acids in length, about10 to 65 amino acids in length, about 10 to 60 amino acids in length,about 10 to 55 amino acids in length, about 10 to 50 amino acids inlength, about 10 to 45 amino acids in length, about 10 to 40 amino acidsin length, about 10 to 35 amino acids in length, about 10 to 30 aminoacids in length, about 10 to 25 amino acids in length, about 10 to 20amino acids in length, about 10 to 15 amino acids in length, about 20 to30 amino acids in length, about 30 to 40 amino acids in length, about 40to 50 amino acids in length, about 50 to 60 amino acids in length, about60 to 70 amino acids in length, about 70 to 80 amino acids in length,about 80 to 90 amino acids in length, about 90 to 100 amino acids inlength, about 20 to 90 amino acids in length, about 30 to 80 amino acidsin length, about 40 to 70 amino acids in length, about 50 to 60 aminoacids in length, or any range in between. In some embodiments, a peptidetag is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 amino acids in length.

Peptide tags included in a first chimeric polypeptide of a multi-chainchimeric polypeptide, a second chimeric polypeptide of the multi-chainchimeric polypeptide, or both can be of any suitable length. Forexample, peptide tags can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or more amino acids in length. In embodiments in which amulti-chain chimeric polypeptide includes two or more peptide tags, thetwo or more peptide tags can be of the same or different lengths. Insome embodiments, any of the peptide tags disclosed herein may includeone or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, ormore amino acids) at the N-terminus and/or C-terminus, so long as thefunction of the peptide tag remains intact. For example, a myc taghaving the amino acid sequence EQKLISEEDL (SEQ ID NO: 64) can includeone or more additional amino acids (e.g., at the N-terminus and/or theC-terminus of the peptide tag), while still retaining the ability to bebound by an antibody (e.g., 9E10).

Exemplary Multi-Chain Chimeric Polypeptides—Type A

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor of IL-18 or a receptor of IL-12. In some examples of thesemulti-chain chimeric polypeptides, the first target-binding domain andthe soluble tissue factor domain directly abut each other in the firstchimeric polypeptide. In some examples of these multi-chain chimericpolypeptides, the first chimeric polypeptide further comprises a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe first target-binding domain and the soluble tissue factor domain inthe first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one orboth of the first target-binding domain and the second target-bindingdomain is an agonistic antigen-binding domain. In some embodiments ofthese multi-chain chimeric polypeptides, the first target-binding domainand the second target-binding domain are each agonistic antigen-bindingdomains. In some embodiments of these multi-chain chimeric polypeptides,the antigen-binding domain includes a scFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one orboth of the first target-binding domain and the second target-bindingdomain is a soluble IL-15 or a soluble IL-18. In some embodiments ofthese multi-chain chimeric polypeptides, the first target-binding domainand the second target-binding domain are each independently a solubleIL-15 or a soluble IL-18. In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain and the secondtarget-binding domain both bind specifically to a receptor of IL-18 or areceptor of IL-12. In some embodiments of these multi-chain chimericpolypeptides, the first target-binding domain and the secondtarget-binding domain bind specifically to the same epitope. In someembodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the second target-binding domain include thesame amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to a receptor for IL-12,and the second target-binding domain binds specifically to a receptorfor IL-18. In some embodiments of these multi-chain chimericpolypeptides, the first target-binding domain binds specifically to areceptor for IL-18, and the second target-binding domain bindspecifically to a receptor for IL-12.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain includes a soluble IL-18 (e.g., a solublehuman IL-18).

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-18 includes a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 16) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTVQNED.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-18 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 65) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain includes a soluble IL-12 (e.g., a solublehuman IL-12). In some embodiments of these multi-chain chimericpolypeptides, the soluble human IL-15 includes a sequence of solublehuman IL-12β (p40) and a sequence of soluble human IL-12α (p35). In someembodiments of these multi-chain chimeric polypeptides, the solubleIL-15 human IL-15 further includes a linker sequence (e.g., any of theexemplary linker sequences described herein) between the sequence ofsoluble IL-12β (p40) and the sequence of soluble human IL-12α (p35). Insome examples of these multi-chain chimeric polypeptides, the linkersequence comprises GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, thesequence of soluble human IL-12β (p40) comprises a sequence that is atleast 80% identical (e.g., at least 82% identical, at least 84%identical, at least 86% identical, at least 88% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 66) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEW ASVPCS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-12β (p40) is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 67) ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGG GCCAGCGTGCCTTGTTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-12α (p35) includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 68) RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-12α (p35) is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 69) CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 70) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 71) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCC AGATGTTCATCAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 72) MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHI VQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 73) ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGCTACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 74) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 75) ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 76) MKWVTFISLLFLFSSAYSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCI R.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 77) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATC CGG.Exemplary Multi-Chain Chimeric Polypeptides—Type B

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor of IL-21 or to TGF-β. In some examples of these multi-chainchimeric polypeptides, the first target-binding domain and the solubletissue factor domain directly abut each other in the first chimericpolypeptide. In some examples of these multi-chain chimericpolypeptides, the first chimeric polypeptide further comprises a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe first target-binding domain and the soluble tissue factor domain inthe first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one orboth of the first target-binding domain and the second target-bindingdomain is a soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or asoluble TGF-β receptor (e.g., a soluble TGFRβRII receptor). In someembodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the second target-binding domain are eachindependently a soluble IL-21 or a soluble TGF-β receptor (e.g., asoluble TGFRβRII receptor). In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain and the secondtarget-binding domain both bind specifically to a receptor of IL-21 orto TGF-β. In some embodiments of these multi-chain chimericpolypeptides, the first target-binding domain and the secondtarget-binding domain bind specifically to the same epitope. In someembodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the second target-binding domain include thesame amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to a receptor for IL-21,and the second target-binding domain binds specifically to TGF-β. Insome embodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain binds specifically to TGF-β, and the secondtarget-binding domain bind specifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain includes a soluble IL-21 (e.g., a solublehuman IL-21). In some embodiments of these multi-chain chimericpolypeptides, the soluble human IL-21 includes a sequence that is atleast 80% identical (e.g., at least 82% identical, at least 84%identical, at least 86% identical, at least 88% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 78) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 79) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain includes a soluble TGF-β receptor (e.g., asoluble TGFRβRII receptor (e.g., a soluble human TGFRβRII receptor)). Insome embodiments of these multi-chain chimeric polypeptides, the solublehuman TGFRβRII includes a first sequence of soluble human TGFRβRII and asecond sequence of soluble human TGFRβRII. In some embodiments of thesemulti-chain chimeric polypeptides, the soluble human TGFRβRII includes alinker disposed between the first sequence of soluble human TGFRβRII andthe second sequence of soluble human TGFRβRII. In some examples of thesemulti-chain chimeric polypeptides, the linker includes the sequenceGGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 80) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 81) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 82) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 83) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human TGFRβRII receptor is encoded by a sequence that is atleast 80% identical (e.g., at least 82% identical, at least 84%identical, at least 86% identical, at least 88% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 84) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thehuman TGFβRII receptor includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 85) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 86) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 87) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 88) MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 89) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCT CC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 90) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKC IR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 91) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGT ATTAGA.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 92) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 93) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAA ATGTATTAGA.Exemplary Multi-Chain Chimeric Polypeptides—Type C

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor of IL-7 or a receptor of IL-21. In some examples of thesemulti-chain chimeric polypeptides, the first target-binding domain andthe soluble tissue factor domain directly abut each other in the firstchimeric polypeptide. In some examples of these multi-chain chimericpolypeptides, the first chimeric polypeptide further comprises a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe first target-binding domain and the soluble tissue factor domain inthe first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one orboth of the first target-binding domain and the second target-bindingdomain is a soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or asoluble IL-7 (e.g., a soluble human IL-7 polypeptide). In someembodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the second target-binding domain are eachindependently a soluble IL-21 or a soluble IL-7. In some embodiments ofthese multi-chain chimeric polypeptides, the first target-binding domainand the second target-binding domain both bind specifically to areceptor of IL-21 or a receptor of IL-7. In some embodiments of thesemulti-chain chimeric polypeptides, the first target-binding domain andthe second target-binding domain bind specifically to the same epitope.In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain and the second target-binding domain includethe same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to a receptor for IL-21,and the second target-binding domain binds specifically to a receptorfor IL-7. In some embodiments of these multi-chain chimericpolypeptides, the first target-binding domain binds specifically to areceptor for IL-7, and the second target-binding domain bindsspecifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain includes a soluble IL-21 (e.g., a solublehuman IL-21).

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 includes a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 78) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 94) CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 79) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesequence of soluble human IL-7 comprises a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 11) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-7 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 95) GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAA GAACAC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 96) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 97) CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 98) MGVKVLFALICIAVAEAQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 99) ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 100) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLN KATNVAHWTTPSLKCIR

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 101) GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAG A.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 102) MGVKVLFALICIAVAEADCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 103) ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTA GA.Exemplary Multi-Chain Chimeric Polypeptides—Type D

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor of IL-7 or a receptor of IL-21. In some examples of thesemulti-chain chimeric polypeptides, the first target-binding domain andthe soluble tissue factor domain directly abut each other in the firstchimeric polypeptide. In some examples of these multi-chain chimericpolypeptides, the first chimeric polypeptide further comprises a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe first target-binding domain and the soluble tissue factor domain inthe first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein. In someembodiments of these multi-chain chimeric polypeptides, one or both ofthe first target-binding domain and the second target-binding domain isa soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or a solubleIL-7 (e.g., a soluble human IL-7 polypeptide). In some embodiments ofthese multi-chain chimeric polypeptides, the first target-binding domainand the second target-binding domain are each independently a solubleIL-21 or a soluble IL-7. In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain and the secondtarget-binding domain both bind specifically to a receptor of IL-21 or areceptor of IL-7. In some embodiments of these multi-chain chimericpolypeptides, the first target-binding domain and the secondtarget-binding domain bind specifically to the same epitope. In someembodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the second target-binding domain include thesame amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to a receptor for IL-21,and the second target-binding domain binds specifically to a receptorfor IL-7. In some embodiments of these multi-chain chimericpolypeptides, the first target-binding domain binds specifically to areceptor for IL-7, and the second target-binding domain bindsspecifically to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 includes a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 78) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 94) CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 79) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesequence of soluble human IL-7 comprises a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 11) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-7 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 95) GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAA GAACAC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 104) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 105) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATA CCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 106) MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI NTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 107) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATC AATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 108) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 109) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 110) MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 111) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG.Exemplary Multi-Chain Chimeric Polypeptides—Type E

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor for IL-18 (e.g., a soluble human IL-18), a receptor for IL-12(e.g., a soluble human IL-12), or CD16 (e.g., an anti-CD16 scFv). Insome embodiments of these multi-chain chimeric polypeptides describedherein, the first chimeric polypeptide further includes the additionaltarget-binding domain. In some embodiments of these multi-chain chimericpolypeptides described herein, the second chimeric polypeptide furtherincludes the additional target-binding domain. In some embodiments ofthese multi-chain chimeric polypeptides described herein, the additionaltarget-binding domain binds specifically to CD16 or a receptor forIL-12.

In some examples of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide. In some examples of thesemulti-chain chimeric polypeptides, the first chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersdescribed herein) between the first target-binding domain and thesoluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second domain of the pair ofaffinity domains directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the additionaltarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second target-binding domaindirectly abut each other in the second chimeric polypeptide. In someembodiments of these multi-chain chimeric polypeptides, the secondchimeric polypeptide further includes a linker sequence (e.g., any ofthe exemplary linkers described herein) between the secondtarget-binding domain and the additional target-binding domain in thesecond chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one ormore of the first target-binding domain, the second target-bindingdomain and the additional antigen-binding domain is an agonisticantigen-binding domain. In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain, the secondtarget-binding domain, and the additional antigen-binding domain areeach agonistic antigen-binding domains. In some embodiments of thesemulti-chain chimeric polypeptides, the antigen-binding domain includes ascFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one orboth of the first target-binding domain and the second target-bindingdomain is a soluble IL-15 or a soluble IL-18. In some embodiments ofthese multi-chain chimeric polypeptides, the first target-binding domainand the second target-binding domain are each independently a solubleIL-15 or a soluble IL-18. In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain and the secondtarget-binding domain both bind specifically to a receptor of IL-18 or areceptor of IL-12. In some embodiments of these multi-chain chimericpolypeptides, the first target-binding domain and the secondtarget-binding domain bind specifically to the same epitope. In someembodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the second target-binding domain include thesame amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to a receptor for IL-12,and the second target-binding domain binds specifically to a receptorfor IL-18. In some embodiments of these multi-chain chimericpolypeptides, the first target-binding domain binds specifically to areceptor for IL-18, and the second target-binding domain bindspecifically to a receptor for IL-12. In some embodiments of thesemulti-chain chimeric polypeptides, the first target-binding domain bindsspecifically to CD16, and the second target-binding domain bindsspecifically to a receptor for IL-18. In some embodiments of thesemulti-chain chimeric polypeptides, the first target-binding domain bindsspecifically to a receptor for IL-18, and the second target-bindingdomain bind specifically to CD16.

In some embodiments of these multi-chain chimeric polypeptides, two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same antigen. In some embodiments, two or more ofthe first target-binding domain, the second target-binding domain, andthe one or more additional target-binding domains bind specifically tothe same epitope. In some embodiments, two or more of the firsttarget-binding domain, the second target-binding domain, and the one ormore additional target-binding domains comprise the same amino acidsequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain includes a soluble IL-18 (e.g., a solublehuman IL-18).

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-18 includes a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 16) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTVQNED.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-18 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 65) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain includes a soluble IL-12 (e.g., a solublehuman IL-12). In some embodiments of these multi-chain chimericpolypeptides, the soluble human IL-15 includes a sequence of solublehuman IL-12β (p40) and a sequence of soluble human IL-12α (p35). In someembodiments of these multi-chain chimeric polypeptides, the solubleIL-15 (e.g., soluble human IL-15) further includes a linker sequence(e.g., any of the exemplary linker sequences described herein) betweenthe sequence of soluble IL-12β (p40) and the sequence of soluble humanIL-12α (p35). In some examples of these multi-chain chimericpolypeptides, the linker sequence comprises GGGGSGGGGSGGGGS (SEQ ID NO:7).

In some embodiments of these multi-chain chimeric polypeptides, thesequence of soluble human IL-12β (p40) comprises a sequence that is atleast 80% identical (e.g., at least 82% identical, at least 84%identical, at least 86% identical, at least 88% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 66) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEW ASVPCS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-12β (p40) is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 67) ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGG GCCAGCGTGCCTTGTTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-12α (p35) includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 68) RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-12α (p35) is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 69) CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain includes an scFv that specificallybinds to CD16 (e.g., an anti-CD16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 includes a light chain variable domainthat includes a sequence that is at least 80% identical (e.g., at least82% identical, at least 84% identical, at least 86% identical, at least88% identical, at least 90% identical, at least 92% identical, at least94% identical, at least 96% identical, at least 98% identical, at least99% identical, or 100% identical) to:

(SEQ ID NO: 112) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGG GTKLTVGH.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 is encoded by a light chain variabledomain sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 113) TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 includes a heavy chain variable domainthat includes a sequence that is at least 80% identical (e.g., at least82% identical, at least 84% identical, at least 86% identical, at least88% identical, at least 90% identical, at least 92% identical, at least94% identical, at least 96% identical, at least 98% identical, at least99% identical, or 100% identical) to:

(SEQ ID NO: 114) EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGR SLLFDYWGQGTLVTVSR.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 is encoded by a heavy chain variabledomain sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 115) GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAG G.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 116) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 117) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCC AGATGTTCATCAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 118) MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHI VQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 119) ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGCTACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 120) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 121) ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCAC CCTGGTGACCGTGTCCAGG.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 122) MKWVTFISLLFLFSSAYSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 123) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG.Exemplary Multi-Chain Chimeric Polypeptides—Type F

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor for IL-7 (e.g., a soluble human IL-7), CD16 (e.g., an anti-CD16scFv), or a receptor for IL-21 (e.g., a soluble human IL-21). In someembodiments of these multi-chain chimeric polypeptides described herein,the first chimeric polypeptide further includes the additionaltarget-binding domain. In some embodiments of these multi-chain chimericpolypeptides described herein, the second chimeric polypeptide furtherincludes the additional target-binding domain. In some embodiments ofthese multi-chain chimeric polypeptides described herein, the additionaltarget-binding domain binds specifically to CD16 or a receptor forIL-21.

In some examples of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide. In some examples of thesemulti-chain chimeric polypeptides, the first chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersdescribed herein) between the first target-binding domain and thesoluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second domain of the pair ofaffinity domains directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the additionaltarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second target-binding domaindirectly abut each other in the second chimeric polypeptide. In someembodiments of these multi-chain chimeric polypeptides, the secondchimeric polypeptide further includes a linker sequence (e.g., any ofthe exemplary linkers described herein) between the secondtarget-binding domain and the additional target-binding domain in thesecond chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one ormore of the first target-binding domain, the second target-bindingdomain and the additional antigen-binding domain is an agonisticantigen-binding domain. In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain, the secondtarget-binding domain, and the additional antigen-binding domain areeach agonistic antigen-binding domains. In some embodiments of thesemulti-chain chimeric polypeptides, the antigen-binding domain includes ascFv or single-domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain binds specifically toa receptor IL-7 and the second target-binding domain binds specificallyto CD16 or a receptor for IL-21. In some embodiments of any of themulti-chain chimeric polypeptides described herein, the firsttarget-binding domain includes a soluble IL-7 protein. In someembodiments of any of the multi-chain chimeric polypeptides describedherein, the soluble IL-7 protein is a soluble human IL-7. In someembodiments of any of the multi-chain chimeric polypeptides describedherein, the second antigen-binding domain includes a target-bindingdomain that binds specifically to CD16. In some embodiments of any ofthe multi-chain chimeric polypeptides described herein, the secondtarget-binding domain includes an scFv that binds specifically to CD16.In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the second target-binding domain binds specifically toa receptor for IL-21. In some embodiments of any of the multi-chainchimeric polypeptides described herein, the second target-binding domainincludes a soluble IL-21. In some embodiments of any of the multi-chainchimeric polypeptides described herein, the soluble IL-21 is a solublehuman IL-21. In some embodiments of any of the multi-chain chimericpolypeptides described herein, the second chimeric polypeptide furtherincludes an additional target-binding domain that binds specifically toa receptor for IL-21. In some embodiments of any of the multi-chainchimeric polypeptides described herein, the additional target-bindingdomain includes a soluble IL-21. In some embodiments of any of themulti-chain chimeric polypeptides described herein, the soluble IL-21 isa soluble human IL-21. In some embodiments of any of the multi-chainchimeric polypeptides described herein, the second chimeric polypeptidefurther includes an additional target-binding domain that bindsspecifically to CD16.

In some embodiments of these multi-chain chimeric polypeptides, two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same antigen. In some embodiments, two or more ofthe first target-binding domain, the second target-binding domain, andthe one or more additional target-binding domains bind specifically tothe same epitope. In some embodiments, two or more of the firsttarget-binding domain, the second target-binding domain, and the one ormore additional target-binding domains comprise the same amino acidsequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain includes a soluble IL-7 (e.g., a solublehuman IL-7).

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-7 includes a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 11) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-7 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 95) GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAA GAACAC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-7 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 124) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT.

In some embodiments of these multi-chain chimeric polypeptides, thesequence of soluble human IL-21 comprises a sequence that is at least80% identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 78) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 94) CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 79) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain includes an scFv that specificallybinds to CD16 (e.g., an anti-CD16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 includes a light chain variable domainthat includes a sequence that is at least 80% identical (e.g., at least82% identical, at least 84% identical, at least 86% identical, at least88% identical, at least 90% identical, at least 92% identical, at least94% identical, at least 96% identical, at least 98% identical, at least99% identical, or 100% identical) to:

(SEQ ID NO: 112) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGG GTKLTVGH.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 is encoded by a light chain variabledomain sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 113) TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 includes a heavy chain variable domainthat includes a sequence that is at least 80% identical (e.g., at least82% identical, at least 84% identical, at least 86% identical, at least88% identical, at least 90% identical, at least 92% identical, at least94% identical, at least 96% identical, at least 98% identical, at least99% identical, or 100% identical) to:

(SEQ ID NO: 114) EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGR SLLFDYWGQGTLVTVSR.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 is encoded by a heavy chain variabledomain sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 115) GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAG G.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 125) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 126) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATA CCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 127) MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI NTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 128) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATC AATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 129) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 130) TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACG GCTCCGAGGACTCC.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 131) MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRT HGSEDS.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 132) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACC CACGGCTCCGAGGACTCC.Exemplary Multi-Chain Chimeric Polypeptides—Type G

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to TGFβ(e.g., a human TGFβRII receptor), CD16 (e.g., an anti-CD16 scFv), or areceptor for IL-21 (e.g., a soluble human IL-21). In some embodiments ofthese multi-chain chimeric polypeptides described herein, the firstchimeric polypeptide further includes the additional target-bindingdomain. In some embodiments of these multi-chain chimeric polypeptidesdescribed herein, the second chimeric polypeptide further includes theadditional target-binding domain. In some embodiments of thesemulti-chain chimeric polypeptides described herein, the additionaltarget-binding domain binds specifically to CD16 or a receptor forIL-21.

In some examples of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide. In some examples of thesemulti-chain chimeric polypeptides, the first chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersdescribed herein) between the first target-binding domain and thesoluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second domain of the pair ofaffinity domains directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the additionaltarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second target-binding domaindirectly abut each other in the second chimeric polypeptide. In someembodiments of these multi-chain chimeric polypeptides, the secondchimeric polypeptide further includes a linker sequence (e.g., any ofthe exemplary linkers described herein) between the secondtarget-binding domain and the additional target-binding domain in thesecond chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one ormore of the first target-binding domain, the second target-bindingdomain and the additional antigen-binding domain is an agonisticantigen-binding domain. In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain, the secondtarget-binding domain, and the additional antigen-binding domain areeach agonistic antigen-binding domains. In some embodiments of thesemulti-chain chimeric polypeptides, the antigen-binding domain includes ascFv or single-domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to TGF-β,CD16, or a receptor for IL-21. In some embodiments of any of themulti-chain chimeric polypeptides described herein, the firsttarget-binding domain binds specifically to a TGF-β and the secondtarget-binding domain binds specifically to CD16 or a receptor of IL-21.In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain is a soluble TGF-βreceptor. In some embodiments of any of the multi-chain chimericpolypeptides described herein, soluble TGF-β receptor is a solubleTGFβRII receptor. In some embodiments of any of the multi-chain chimericpolypeptides described herein, the second target-binding domain bindsspecifically to CD16. In some embodiments of any of the multi-chainchimeric polypeptides described herein, the second antigen-bindingdomain includes an antigen-binding domain that binds specifically toCD16. In some embodiments of any of the multi-chain chimericpolypeptides described herein, the second antigen-binding domainincludes an scFv that binds specifically to CD16. In some embodiments ofany of the multi-chain chimeric polypeptides described herein, thesecond target-binding domain binds specifically to a receptor for IL-21.In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the second target-binding domain includes a solubleIL-21. In some embodiments of any of the multi-chain chimericpolypeptides described herein, the second target-binding domain includesa soluble human IL-21. In some embodiments of any of the multi-chainchimeric polypeptides described herein, the second chimeric polypeptidefurther includes an additional target-binding domain that bindsspecifically to a receptor for IL-21. In some embodiments of any of themulti-chain chimeric polypeptides described herein, the additionaltarget-binding domain includes a soluble IL-21. In some embodiments ofany of the multi-chain chimeric polypeptides described herein, thesoluble IL-21 is a soluble human IL-21. In some embodiments of any ofthe multi-chain chimeric polypeptides described herein, the secondchimeric polypeptide further includes an additional target-bindingdomain that binds specifically to CD16.

In some embodiments of these multi-chain chimeric polypeptides, two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same antigen. In some embodiments, two or more ofthe first target-binding domain, the second target-binding domain, andthe one or more additional target-binding domains bind specifically tothe same epitope. In some embodiments, two or more of the firsttarget-binding domain, the second target-binding domain, and the one ormore additional target-binding domains comprise the same amino acidsequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain includes a TGFβRII receptor (e.g., a solublehuman TGFβRII receptor). In some embodiments of these multi-chainchimeric polypeptides, the soluble human TGFRβRII includes a firstsequence of soluble human TGFRβRII and a second sequence of solublehuman TGFRβRII. In some embodiments of these multi-chain chimericpolypeptides, the soluble human TGFRβRII includes a linker disposedbetween the first sequence of soluble human TGFRβRII and the secondsequence of soluble human TGFRβRII. In some examples of thesemulti-chain chimeric polypeptides, the linker includes the sequenceGGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 80) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 81) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 82) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 83) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 85) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human TGFβRII receptor is encoded by a sequence that is at least80% identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 84) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesequence of soluble human IL-21 comprises a sequence that is at least80% identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 78) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 94) CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 79) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 includes a light chain variable domainthat includes a sequence that is at least 80% identical (e.g., at least82% identical, at least 84% identical, at least 86% identical, at least88% identical, at least 90% identical, at least 92% identical, at least94% identical, at least 96% identical, at least 98% identical, at least99% identical, or 100% identical) to:

(SEQ ID NO: 112) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGG GTKLTVGH.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 is encoded by a light chain variabledomain sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 113) TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 includes a heavy chain variable domainthat includes a sequence that is at least 80% identical (e.g., at least82% identical, at least 84% identical, at least 86% identical, at least88% identical, at least 90% identical, at least 92% identical, at least94% identical, at least 96% identical, at least 98% identical, at least99% identical, or 100% identical) to:

(SEQ ID NO: 114) EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGR SLLFDYWGQGTLVTVSR.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 is encoded by a heavy chain variabledomain sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 115) GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAG G.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 133) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 134) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCAT CAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 135) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 136) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGT TCATCAATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 137) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 138) TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACG GCTCCGAGGACTCC.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 139) MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRT HGSEDS.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 140) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACC CACGGCTCCGAGGACTCC.Exemplary Multi-Chain Chimeric Polypeptides—Type H

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor of IL-7. In some examples of these multi-chain chimericpolypeptides, the first target-binding domain and the soluble tissuefactor domain directly abut each other in the first chimericpolypeptide. In some examples of these multi-chain chimericpolypeptides, the first chimeric polypeptide further comprises a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe first target-binding domain and the soluble tissue factor domain inthe first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain and the second target-binding domain eachindependently bind specifically to a receptor for IL-7. In someembodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the second target-binding domain bindspecifically to the same epitope. In some embodiments of thesemulti-chain chimeric polypeptides, the first target-binding domain andthe second target-binding domain include the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain and the second target-binding domain includea soluble IL-7 (e.g., a soluble human IL-7). In some embodiments ofthese multi-chain chimeric polypeptides, the soluble human IL-7 includesa sequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 11) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-7 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 95) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 141) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 142) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATA CCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 143) MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI NTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 144) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATC AATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 145) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLN KATNVAHWTTPSLKCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 146) GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAG A.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 147) MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 148) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCA TCCGG.Exemplary Multi-Chain Chimeric Polypeptides—Type I

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to TGF-β.In some examples of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide. In some examples of thesemulti-chain chimeric polypeptides, the first chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersdescribed herein) between the first target-binding domain and thesoluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain and the second target-binding domain eachindependently bind specifically to TGF-β. In some embodiments of thesemulti-chain chimeric polypeptides, the first target-binding domain andthe second target-binding domain bind specifically to the same epitope.In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain and the second target-binding domain includethe same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain and the second target-binding domain is asoluble TGF-β receptor (e.g., a soluble TGFβRII receptor, e.g., asoluble human TGFβRII). In some embodiments of these multi-chainchimeric polypeptides, the soluble human TGFRβRII includes a firstsequence of soluble human TGFRβRII and a second sequence of solublehuman TGFRβRII. In some embodiments of these multi-chain chimericpolypeptides, the soluble human TGFRβRII includes a linker disposedbetween the first sequence of soluble human TGFRβRII and the secondsequence of soluble human TGFRβRII. In some examples of thesemulti-chain chimeric polypeptides, the linker includes the sequenceGGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 80) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to: c (SEQ ID NO: 81).

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 82) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 83) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 85) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 84) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 149) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 150) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCAT CAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 151) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 152) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGT TCATCAATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 153) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKC IR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 154) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGC ATCCGG.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 155) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 156) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAA GTGCATCCGG.Exemplary Multi-Chain Chimeric Polypeptides—Type J

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor of IL-7, a receptor of IL-21, or a receptor of CD137L. In someembodiments of these multi-chain chimeric polypeptides described herein,the second chimeric polypeptide further includes the additionaltarget-binding domain. In some embodiments of these multi-chain chimericpolypeptides described herein, the additional target-binding domainbinds specifically to a receptor for IL-21 (e.g., a soluble IL-21, e.g.,a soluble human IL-21) or a receptor for CD137L (e.g., a soluble CD137L,e.g., a soluble human CD137L).

In some examples of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide. In some examples of thesemulti-chain chimeric polypeptides, the first chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersdescribed herein) between the first target-binding domain and thesoluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second domain of the pair ofaffinity domains directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the additionaltarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second target-binding domaindirectly abut each other in the second chimeric polypeptide. In someembodiments of these multi-chain chimeric polypeptides, the secondchimeric polypeptide further includes a linker sequence (e.g., any ofthe exemplary linkers described herein) between the secondtarget-binding domain and the additional target-binding domain in thesecond chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments, the second chimeric polypeptide can include anadditional target-binding domain. In some embodiments, the additionaltarget-binding domain and the

In some embodiments of these multi-chain chimeric polypeptides, one ormore of the first target-binding domain, the second target-bindingdomain and the additional target-binding domain is an agonisticantigen-binding domain. In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain, the secondtarget-binding domain, and the additional target-binding domain are eachagonistic antigen-binding domains. In some embodiments of thesemulti-chain chimeric polypeptides, the antigen-binding domain includes ascFv or single-domain antibody. In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain bindsspecifically to a receptor for IL-7, and the second target-bindingdomain binds specifically to a receptor for IL-21 or a receptor forCD137L. In some embodiments, the additional target-binding domain bindsspecifically to a receptor for IL-21 or a receptor for CD137L.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain is a soluble IL-7 (e.g., a soluble humanIL-7). In some embodiments of these multi-chain chimeric polypeptides,the soluble human IL-7 includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 11) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-7 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 124) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain or the additional target-binding domainbinds specifically to a receptor for IL-21. In some embodiments of thesemulti-chain chimeric polypeptides, the second target-binding domain orthe additional target-binding domain is a soluble IL-21 (e.g., a solublehuman IL-21).

In some embodiments of these multi-chain chimeric polypeptides, asoluble human IL-21 includes a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 78) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments of these multi-chain chimeric polypeptides, asoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 79) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain binds specifically to a receptor forCD137L. In some embodiments of these multi-chain chimeric polypeptides,the second chimeric polypeptide further comprises an additionaltarget-binding domain that binds specifically to a receptor for CD137L.In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain and/or the additional target-binding domainis a soluble CD137L (e.g., a soluble human CD137L).

In some embodiments of these multi-chain chimeric polypeptides, asoluble human CD137L includes a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 157) REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE.

In some embodiments of these multi-chain chimeric polypeptides, asoluble human CD137L is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 158) CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGG AA.

In some embodiments of these multi-chain chimeric polypeptides, asoluble human CD137L includes a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 159) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT QGATVLGLFRVTPEI.

In some embodiments of these multi-chain chimeric polypeptides, asoluble human CD137L is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 160) GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 161) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 162) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATA CCTCC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 163) MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI NTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 164) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATC AATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 165) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 166) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 167) MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFR VTPEIPAGLPSPRSE.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 168) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 169) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 170) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 171) MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 172) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC.Exemplary Multi-Chain Chimeric Polypeptides—Type K

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor of IL-7 or TGF-β. In some examples of these multi-chainchimeric polypeptides, the first target-binding domain and the solubletissue factor domain directly abut each other in the first chimericpolypeptide. In some examples of these multi-chain chimericpolypeptides, the first chimeric polypeptide further comprises a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe first target-binding domain and the soluble tissue factor domain inthe first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to a receptor for IL-7,and the second target-binding domain binds specifically to TGF-β. Insome embodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain binds specifically to TGF-β, and the secondtarget-binding domain binds specifically to a receptor for IL-7.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain includes a soluble IL-7 protein (e.g., asoluble human IL-7 protein). In some embodiments of these multi-chainchimeric polypeptides, the soluble human IL-7 protein includes asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 11) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMG TKEH.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-7 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 124) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain comprises a target-binding domain thatbinds specifically to TGF-β. In some embodiments of these multi-chainchimeric polypeptides, the second target-binding domain is a solubleTGF-β receptor (e.g., a soluble TGFβRII receptor, e.g., a soluble humanTGFβRII receptor). In some embodiments of these multi-chain chimericpolypeptides, the soluble human TGFRβRII includes a first sequence ofsoluble human TGFRβRII and a second sequence of soluble human TGFRβRII.In some embodiments of these multi-chain chimeric polypeptides, thesoluble human TGFRβRII includes a linker disposed between the firstsequence of soluble human TGFRβRII and the second sequence of solublehuman TGFRβRII. In some examples of these multi-chain chimericpolypeptides, the linker includes the sequence GGGGSGGGGSGGGGS (SEQ IDNO: 7).

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 80) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 81) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 82) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 83) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 85) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 84) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 173) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 174) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATA CCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 175) MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFI NTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 176) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATC AATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 177) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSL KCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 178) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGC ATCCGG.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 179) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPSLKCIR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 180) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAA GTGCATCCGG.Exemplary Multi-Chain Chimeric Polypeptides—Type L

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to TGF-β,a receptor of IL-21, or a receptor of CD137L. In some embodiments ofthese multi-chain chimeric polypeptides described herein, the secondchimeric polypeptide further includes the additional target-bindingdomain. In some embodiments of these multi-chain chimeric polypeptidesdescribed herein, the additional target-binding domain bindsspecifically to a receptor for IL-21 (e.g., a soluble IL-21, e.g., asoluble human IL-21) or a receptor for CD137L (e.g., a soluble CD137L,e.g., a soluble human CD137L).

In some examples of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide. In some examples of thesemulti-chain chimeric polypeptides, the first chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersdescribed herein) between the first target-binding domain and thesoluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second domain of the pair ofaffinity domains directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the additionaltarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second target-binding domaindirectly abut each other in the second chimeric polypeptide. In someembodiments of these multi-chain chimeric polypeptides, the secondchimeric polypeptide further includes a linker sequence (e.g., any ofthe exemplary linkers described herein) between the secondtarget-binding domain and the additional target-binding domain in thesecond chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one ormore of the first target-binding domain, the second target-bindingdomain and the additional target-binding domain is an agonisticantigen-binding domain. In some embodiments of these multi-chainchimeric polypeptides, the first target-binding domain, the secondtarget-binding domain, and the additional target-binding domain are eachagonistic antigen-binding domains. In some embodiments of thesemulti-chain chimeric polypeptides, the antigen-binding domain includes ascFv or single-domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to TGF-β and the secondtarget-binding domain binds specifically to a receptor for IL-21 or areceptor for CD137L.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain is a soluble TGF-β receptor (e.g., a solubleTGFβRII receptor, e.g., a soluble human TGFβRII receptor). In someembodiments of these multi-chain chimeric polypeptides, the solublehuman TGFRβRII includes a first sequence of soluble human TGFRβRII and asecond sequence of soluble human TGFRβRII. In some embodiments of thesemulti-chain chimeric polypeptides, the soluble human TGFRβRII includes alinker disposed between the first sequence of soluble human TGFRβRII andthe second sequence of soluble human TGFRβRII. In some examples of thesemulti-chain chimeric polypeptides, the linker includes the sequenceGGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 80) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 81) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 82) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 83) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 85) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 84) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain or the additional target-binding domainbinds specifically to a receptor for IL-21. In some embodiments of thesemulti-chain chimeric polypeptides, the second target-binding domain orthe additional target-binding domain includes a soluble IL-21 (e.g., asoluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, asoluble human IL-21 includes a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 78) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human IL-21 is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 79) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain or the additional target-binding domainbinds specifically to a receptor for CD137L. In some embodiments ofthese multi-chain chimeric polypeptides, the second target-bindingdomain and/or the additional target-binding domain includes a solubleCD137L (e.g., a soluble human CD137L).

In some embodiments of these multi-chain chimeric polypeptides, asoluble CD137L includes a sequence that is at least 80% identical (e.g.,at least 82% identical, at least 84% identical, at least 86% identical,at least 88% identical, at least 90% identical, at least 92% identical,at least 94% identical, at least 96% identical, at least 98% identical,at least 99% identical, or 100% identical) to:

(SEQ ID NO: 157) REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE.

In some embodiments of these multi-chain chimeric polypeptides, asoluble CD137L is encoded by a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 158) CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGG AA.

In some embodiments of these multi-chain chimeric polypeptides, asoluble human CD137L includes a sequence that is at least 80% identical(e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical) to:

(SEQ ID NO: 159) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT QGATVLGLFRVTPEI.

In some embodiments of these multi-chain chimeric polypeptides, asoluble human CD137L is encoded by a sequence that is at least 80%identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% identical) to:

(SEQ ID NO: 160) GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 181) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 182) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCAT CAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 183) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 184) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGT TCATCAATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 185) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 186) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 187) MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFR VTPEIPAGLPSPRSE.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 188) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 189) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 190) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 191) MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 192) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC.Exemplary Multi-Chain Chimeric Polypeptides—Type M

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to TGF-βor a receptor of IL-21. In some embodiments of these multi-chainchimeric polypeptides described herein, the second chimeric polypeptidefurther includes the additional target-binding domain. In someembodiments of these multi-chain chimeric polypeptides described herein,the additional target-binding domain binds specifically to a receptorfor IL-21 (e.g., a soluble IL-21, e.g., a soluble human IL-21) or aTGF-β (e.g., a soluble TGF-β receptor, e.g., a soluble TGFβRIIreceptor).

In some examples of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide. In some examples of thesemulti-chain chimeric polypeptides, the first chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersdescribed herein) between the first target-binding domain and thesoluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second domain of the pair ofaffinity domains directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the additionaltarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second target-binding domaindirectly abut each other in the second chimeric polypeptide. In someembodiments of these multi-chain chimeric polypeptides, the secondchimeric polypeptide further includes a linker sequence (e.g., any ofthe exemplary linkers described herein) between the secondtarget-binding domain and the additional target-binding domain in thesecond chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to TGF-β, and the secondtarget-binding domain binds specifically to TGF-β or a receptor forIL-21. In some embodiments of these multi-chain chimeric polypeptides,the first target-binding domain is a soluble TGF-β receptor (e.g., asoluble TGFβRII receptor, e.g., a soluble human TGFβRII receptor). Insome embodiments of these multi-chain chimeric polypeptides, the solublehuman TGFRβRII includes a first sequence of soluble human TGFRβRII and asecond sequence of soluble human TGFRβRII. In some embodiments of thesemulti-chain chimeric polypeptides, the soluble human TGFRβRII includes alinker disposed between the first sequence of soluble human TGFRβRII andthe second sequence of soluble human TGFRβRII. In some examples of thesemulti-chain chimeric polypeptides, the linker includes the sequenceGGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 80) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 81) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 82) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 83) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 85) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 84) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain binds specifically to a receptor for IL-21.In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain includes a soluble IL-21 (e.g., a humansoluble IL-21). In some embodiments of these multi-chain chimericpolypeptides, the soluble IL-21 includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 78) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble IL-21 is encoded by a sequence that is at least 80% identical(e.g., at least 82% identical, at least 84% identical, at least 86%identical, at least 88% identical, at least 90% identical, at least 92%identical, at least 94% identical, at least 96% identical, at least 98%identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 79) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 193) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 194) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCAT CAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 195) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 196) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGT TCATCAATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 197) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 198) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGG ACTCC.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 199) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGS EDS.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 200) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCC GAGGACTCC.Exemplary Multi-Chain Chimeric Polypeptides—Type N

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to TGF-βor CD16. In some embodiments of these multi-chain chimeric polypeptidesdescribed herein, the second chimeric polypeptide further includes theadditional target-binding domain. In some embodiments of thesemulti-chain chimeric polypeptides described herein, the additionaltarget-binding domain binds specifically to CD16 (e.g., an anti-CD16scFv) or a TGF-β (e.g., a soluble TGF-β receptor, e.g., a solubleTGFβRII receptor).

In some examples of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide. In some examples of thesemulti-chain chimeric polypeptides, the first chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersdescribed herein) between the first target-binding domain and thesoluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second domain of the pair ofaffinity domains directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the additionaltarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second target-binding domaindirectly abut each other in the second chimeric polypeptide. In someembodiments of these multi-chain chimeric polypeptides, the secondchimeric polypeptide further includes a linker sequence (e.g., any ofthe exemplary linkers described herein) between the secondtarget-binding domain and the additional target-binding domain in thesecond chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to TGF-β, and the secondtarget-binding domain binds specifically to TGF-β or CD16. In someembodiments of these multi-chain chimeric polypeptides, the firsttarget-binding domain is a soluble TGF-β receptor (e.g., a solubleTGFβRII receptor, e.g., a soluble human TGFβRII receptor). In someembodiments of these multi-chain chimeric polypeptides, the solublehuman TGFRβRII includes a first sequence of soluble human TGFRβRII and asecond sequence of soluble human TGFRβRII. In some embodiments of thesemulti-chain chimeric polypeptides, the soluble human TGFRβRII includes alinker disposed between the first sequence of soluble human TGFRβRII andthe second sequence of soluble human TGFRβRII. In some examples of thesemulti-chain chimeric polypeptides, the linker includes the sequenceGGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 80) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 81) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 82) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 83) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 85) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 84) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain binds specifically to CD16. In someembodiments of these multi-chain chimeric polypeptides, the secondtarget-binding domain includes an anti-CD16 scFv. In some embodiments ofthese multi-chain chimeric polypeptides, the scFv that bindsspecifically to CD16 includes a light chain variable domain thatincludes a sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 112) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGG GTKLTVGH.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 is encoded by a light chain variabledomain sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 113) TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 includes a heavy chain variable domainthat includes a sequence that is at least 80% identical (e.g., at least82% identical, at least 84% identical, at least 86% identical, at least88% identical, at least 90% identical, at least 92% identical, at least94% identical, at least 96% identical, at least 98% identical, at least99% identical, or 100% identical) to:

(SEQ ID NO: 114) EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGR SLLFDYWGQGTLVTVSR.

In some embodiments of these multi-chain chimeric polypeptides, the scFvthat binds specifically to CD16 is encoded by a heavy chain variabledomain sequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least 88%identical, at least 90% identical, at least 92% identical, at least 94%identical, at least 96% identical, at least 98% identical, at least 99%identical, or 100% identical) to:

(SEQ ID NO: 115) GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAG G.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 201) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 202) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCAT CAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 203) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 204) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGT TCATCAATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 205) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 206) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 207) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYW GQGTLVTVSR.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 208) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG.Exemplary Multi-Chain Chimeric Polypeptides—Type O

In some embodiments of any of the multi-chain chimeric polypeptidesdescribed herein, the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to TGF-βor a receptor of CD137L. In some embodiments of these multi-chainchimeric polypeptides described herein, the second chimeric polypeptidefurther includes the additional target-binding domain. In someembodiments of these multi-chain chimeric polypeptides described herein,the additional target-binding domain binds specifically to a receptor toTGF-β (e.g., a soluble TGF-β receptor, e.g., a soluble TGFβRII receptor)or CD137L.

In some examples of these multi-chain chimeric polypeptides, the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide. In some examples of thesemulti-chain chimeric polypeptides, the first chimeric polypeptidefurther comprises a linker sequence (e.g., any of the exemplary linkersdescribed herein) between the first target-binding domain and thesoluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain and the first domain of the pair ofaffinity domains directly abut each other in the first chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the first chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe soluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesecond domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second domain of the pair ofaffinity domains directly abut each other in the second chimericpolypeptide. In some embodiments of these multi-chain chimericpolypeptides, the second chimeric polypeptide further includes a linkersequence (e.g., any of the exemplary linkers described herein) betweenthe second domain of the pair of affinity domains and the additionaltarget-binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, theadditional target-binding domain and the second target-binding domaindirectly abut each other in the second chimeric polypeptide. In someembodiments of these multi-chain chimeric polypeptides, the secondchimeric polypeptide further includes a linker sequence (e.g., any ofthe exemplary linkers described herein) between the secondtarget-binding domain and the additional target-binding domain in thesecond chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble tissue factor domain can be any of the exemplary soluble tissuefactor domains described herein. In some embodiments of thesemulti-chain chimeric polypeptides, the pair of affinity domains can beany of the exemplary pairs of affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, thefirst target-binding domain binds specifically to TGF-β, and the secondtarget-binding domain binds specifically to CD137L. In some embodimentsof these multi-chain chimeric polypeptides, the first target-bindingdomain or the additional target-binding domain is a soluble TGF-βreceptor (e.g., a soluble TGFβRII receptor, e.g., a soluble humanTGFβRII receptor).

In some embodiments of these multi-chain chimeric polypeptides, thesoluble human TGFRβRII includes a first sequence of soluble humanTGFRβRII and a second sequence of soluble human TGFRβRII. In someembodiments of these multi-chain chimeric polypeptides, the solublehuman TGFRβRII includes a linker disposed between the first sequence ofsoluble human TGFRβRII and the second sequence of soluble humanTGFRβRII. In some examples of these multi-chain chimeric polypeptides,the linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 7).

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 80) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor comprises a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 81) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thefirst sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 82) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT.

In some embodiments of these multi-chain chimeric polypeptides, thesecond sequence of soluble human TGFRβRII receptor is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 83) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor includes a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 85) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD.

In some embodiments of these multi-chain chimeric polypeptides, thesoluble TGF-β receptor is encoded by a sequence that is at least 80%identical (e.g., at least 82% identical, at least 84% identical, atleast 86% identical, at least 88% identical, at least 90% identical, atleast 92% identical, at least 94% identical, at least 96% identical, atleast 98% identical, at least 99% identical, or 100% identical) to:

(SEQ ID NO: 84) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC.

In some embodiments of these multi-chain chimeric polypeptides, thesecond target-binding domain includes a soluble CD137L protein (e.g., asoluble human CD137L protein). In some embodiments of these multi-chainchimeric polypeptides, a soluble human CD137L includes a sequence thatis at least 80% identical (e.g., at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical) to:

(SEQ ID NO: 157) REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE.

In some embodiments of these multi-chain chimeric polypeptides, asoluble human CD137L is encoded by a sequence that is at least 80%identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% identical) to:

(SEQ ID NO: 158) CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGG AA.

In some embodiments of these multi-chain chimeric polypeptides, asoluble human CD137L includes a sequence that is at least 80% identical(e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical) to:

(SEQ ID NO: 159) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT QGATVLGLFRVTPEI.

In some embodiments of these multi-chain chimeric polypeptides, asoluble human CD137L is encoded by a sequence that is at least 80%identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% identical) to:

(SEQ ID NO: 160) GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC.

In some embodiments, the first chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 209) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEK NIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 210) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCAT CAATACCTCC.

In some embodiments, a first chimeric polypeptide can include a sequencethat is at least 80% identical (e.g., at least 82% identical, at least84% identical, at least 86% identical, at least 88% identical, at least90% identical, at least 92% identical, at least 94% identical, at least96% identical, at least 98% identical, at least 99% identical, or 100%identical) to:

(SEQ ID NO: 211) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, a first chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 212) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGT TCATCAATACCTCC.

In some embodiments, the second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 213) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRS E.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 214) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCG GAA.

In some embodiments, a second chimeric polypeptide can include asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 215) MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVL GLFRVTPEIPAGLPSPRSE.

In some embodiments, a second chimeric polypeptide is encoded by asequence that is at least 80% identical (e.g., at least 82% identical,at least 84% identical, at least 86% identical, at least 88% identical,at least 90% identical, at least 92% identical, at least 94% identical,at least 96% identical, at least 98% identical, at least 99% identical,or 100% identical) to:

(SEQ ID NO: 216) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAG GTCGGAA.Compositions/Kits

Also provided herein are compositions (e.g., pharmaceuticalcompositions) that include at least one of any multi-chain chimericpolypeptides, any of the cells, or any of the nucleic acids describedherein. In some embodiments, the compositions include at least one ofany of the multi-chain chimeric polypeptides described herein. In someembodiments, the compositions include any of the immune cells (e.g., anyof the immune cells described herein, e.g., any of the immune cellsproduced using any of the methods described herein).

In some embodiments, the pharmaceutical compositions are formulated fordifferent routes of administration (e.g., intravenous, subcutaneous). Insome embodiments, the pharmaceutical compositions can include apharmaceutically acceptable carrier (e.g., phosphate buffered saline).

Single or multiple administrations of pharmaceutical compositions can begiven to a subject in need thereof depending on for example: the dosageand frequency as required and tolerated by the subject. The formulationshould provide a sufficient quantity of active agent to effectivelytreat, prevent or ameliorate conditions, diseases or symptoms. Alsoprovided herein are kits that include any of the multi-chain chimericpolypeptides, compositions, nucleic acids, or cells (e.g., immune cells)described herein. In some embodiments, the kits can include instructionsfor performing any of the methods described herein. In some embodiments,the kits can include at least one dose of any of the pharmaceuticalcompositions described herein.

Nucleic Acids/Vectors

Also provided herein are nucleic acids that encode any of themulti-chain chimeric polypeptides described herein. In some embodiments,a first nucleic acid can encode the first chimeric polypeptide and asecond nucleic acid can encode the second chimeric polypeptide. In someembodiments, a single nucleic acid can encode both the first chimericpolypeptide and the second chimeric polypeptide.

Also provided herein are vectors that include any of the nucleic acidsencoding any of the multi-chain chimeric polypeptides described herein.In some embodiments, a first vector can include a nucleic acid encodingthe first chimeric polypeptide and a second vector can include a nucleicacid encoding the second chimeric polypeptide. In some embodiments, asingle vector can include a first nucleic acid encoding the firstchimeric polypeptide and a second nucleic acid encoding the secondchimeric polypeptide.

Any of the vectors described herein can be an expression vector. Forexample, an expression vector can include a promoter sequence operablylinked to the sequence encoding the first chimeric polypeptide and thesecond chimeric polypeptide.

Non-limiting examples of vectors include plasmids, transposons, cosmids,and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMVvectors), adeno-associated virus (AAV) vectors, lentivirus vectors, andretroviral vectors), and any Gateway® vectors. A vector can, e.g.,include sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host mammalian cell or in an invitro expression system. Skilled practitioners will be capable ofselecting suitable vectors and mammalian cells for making any of themulti-chain chimeric polypeptides described herein.

Cells

Also provided herein are cells (e.g., any of the exemplary cellsdescribed herein or known in the art) comprising any of the nucleicacids described herein that encode any of the multi-chain chimericpolypeptides described herein (e.g., encoding both the first and secondchimeric polypeptides). Also provided herein are cells (e.g., any of theexemplary cells described herein or known in the art) comprising any ofthe nucleic acids described herein that encode any of the first chimericpolypeptides described herein. Also provided are cells (e.g., any of theexemplary cells described herein or known in the art) comprising any ofthe nucleic acids described herein that encode any of the secondchimeric polypeptides described herein.

Also provided herein are cells (e.g., any of the exemplary cellsdescribed herein or known in the art) that include any of the vectorsdescribed herein that encode any of the multi-chain chimericpolypeptides described herein (e.g., encoding both the first and secondchimeric polypeptides). Also provided herein are cells (e.g., any of theexemplary cells described herein or known in the art) that include anyof the vectors described herein that encode any of the first chimericpolypeptides described herein. Also provided herein are cells (e.g., anyof the exemplary cells described herein or known in the art) thatinclude any of the vectors described herein that encode any of thesecond chimeric polypeptides described herein).

In some embodiments of any of the methods described herein, the cell canbe a eukaryotic cell. As used herein, the term “eukaryotic cell” refersto a cell having a distinct, membrane-bound nucleus. Such cells mayinclude, for example, mammalian (e.g., rodent, non-human primate, orhuman), insect, fungal, or plant cells. In some embodiments, theeukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. Insome embodiments, the eukaryotic cell is a higher eukaryote, such asmammalian, avian, plant, or insect cells. Non-limiting examples ofmammalian cells include Chinese hamster ovary cells and human embryonickidney cells (e.g., HEK293 cells).

Methods of introducing nucleic acids and expression vectors into a cell(e.g., a eukaryotic cell) are known in the art. Non-limiting examples ofmethods that can be used to introduce a nucleic acid into a cell includelipofection, transfection, electroporation, microinjection, calciumphosphate transfection, dendrimer-based transfection, cationic polymertransfection, cell squeezing, sonoporation, optical transfection,impalefection, hydrodynamic delivery, magnetofection, viral transduction(e.g., adenoviral and lentiviral transduction), and nanoparticletransfection.

Methods of Producing Multi-Chain Chimeric Polypeptides

Also provided herein are methods of producing any of the multi-chainchimeric polypeptides described herein that include culturing any of thecells described herein in a culture medium under conditions sufficientto result in the production of the multi-chain chimeric polypeptide; andrecovering the multi-chain chimeric polypeptide from the cell and/or theculture medium.

Also provided herein are method of producing any of the multi-chainchimeric polypeptides described herein that include: culturing any ofcells described herein in a first culture medium under conditionssufficient to result in the production of the first chimericpolypeptide; recovering the first chimeric polypeptide from the celland/or the first culture medium; culturing any of the cells describedherein in a second culture medium under conditions sufficient to resultin the production of the second chimeric polypeptide; recovering thesecond chimeric polypeptide from the cell and/or the second culturemedium; and combining (e.g., mixing) the recovered first chimericpolypeptide and the recovered second chimeric polypeptide to form themulti-chain chimeric polypeptide (e.g., any of the multi-chain chimericpolypeptides described herein).

The recovery of the multi-chain chimeric polypeptide, the first chimericpolypeptide, or the second chimeric polypeptide from a cell (e.g., aeukaryotic cell) can be performed using techniques well-known in the art(e.g., ammonium sulfate precipitation, polyethylene glycolprecipitation, ion-exchange chromatography (anion or cation),chromatography based on hydrophobic interaction, metal-affinitychromatography, ligand-affinity chromatography, and size exclusionchromatography).

Methods of culturing cells are well known in the art. Cells can bemaintained in vitro under conditions that favor proliferation,differentiation and growth. Briefly, cells can be cultured by contactinga cell (e.g., any cell) with a cell culture medium that includes thenecessary growth factors and supplements to support cell viability andgrowth.

Also provided herein are multi-chain chimeric polypeptides (e.g., any ofthe multi-chain chimeric polypeptides described herein), first chimericpolypeptides (e.g., any of the first chimeric polypeptides), or secondchimeric polypeptides (e.g., any of the second chimeric polypeptidesdescribed herein) produced by any of the methods described herein.

Methods of Stimulating an Immune Cell

Also provided herein are methods of stimulating an immune cell (e.g.,any of the exemplary immune cells described herein or known in the art)that include contacting an immune cell with an effective amount of anyof the multi-chain chimeric polypeptides described herein or any of thecompositions (e.g., pharmaceutical compositions) described herein. Insome examples, the immune cell is contacted in vitro (e.g., in asuitable liquid culture medium under conditions sufficient to result instimulation of the immune cell).

In some examples, the immune cell has been previously obtained from asubject (e.g., a mammal, e.g., a human). Some embodiments of thesemethods further include obtaining the immune cell from the subject priorto the contacting step.

In some examples, the immune cell is contacted in vivo. In suchembodiments, the multi-chain chimeric polypeptide is administered to asubject (e.g., a mammal, e.g., a human) in an amount sufficient toresult in stimulation of an immune cell in the subject.

In some examples of any of the methods described herein, the immune cellcan be an immature thymocyte, a peripheral blood lymphocyte, a naïve Tcell, a pluripotent Th cell precursor, a lymphoid progenitor cell, aTreg cell, a memory T cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ T cell, atumor-infiltrating T cell, a CD8⁺ T cell, a CD4⁺ T cell, a naturalkiller T cell, a mast cell, a macrophage, a neutrophil, a dendriticcell, a basophil, an eosinophil, or a natural killer cell, or acombination thereof.

In some examples, the immune cell has previously beengenetically-modified to express a chimeric antigen receptor or arecombinant T-cell receptor. In some examples, the immune cell (e.g.,any of the immune cells described herein) has previously beengenetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after thecontacting step, introducing into the immune cell (e.g., any of theimmune cells described herein) a nucleic acid encoding a chimericantigen-receptor or a recombinant T-cell receptor. Some embodiments ofthese methods can further include, after the contacting step,introducing into the immune cell (e.g., any of the immune cellsdescribed herein) a nucleic acid encoding a co-stimulatory molecule(e.g., CD28).

Some embodiments of these methods can further include administering atherapeutically effective amount of the immune cell to a subject in needthereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosedas having an age-related disease or condition. Non-limiting examples ofage-related diseases or disorders include: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identifiedor diagnosed as having a cancer. Non-limiting examples of cancersinclude: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosedor identified as having an infectious disease. Non-limiting examples ofinfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, or influenza virus.

Activation of an immune cell can be determined using methods known inthe art. For example, activation of an immune cell can be determined bydetecting the levels of cytokines and chemokines that are secreted orcytotoxicity granules and regulatory molecules that are upregulated uponactivation of an immune cell. Non-limiting examples of cytokines,chemokines, cytotoxicity granules, and regulatory molecules that aresecreted or upregulated upon activation of an immune cell include: IL-2,IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15,IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFα, granzymes,perforin, TGFβ, STAT3, STAT4, STAT5, RORγT, FOXP3, STAT6, and GATA3. Thedetection of these cytokines, chemokines, cytotoxicity granules, orregulatory molecules can be performed using an immunoassay (e.g., anenzyme-linked immunosorbent assay) and quantitative PCR. For example,activation of an immune cell can result in an increase of about 1% toabout 800% (e.g., about 1% to about 750%, about 1% to about 700%, about1% to about 650%, about 1% to about 600%, about 1% to about 550%, about1% to about 500%, about 1% to about 450%, about 1% to about 400%, about1% to about 350%, about 1% to about 300%, about 1% to about 280%, about1% to about 260%, about 1% to about 240%, about 1% to about 220%, about1% to about 200%, about 1% to about 180%, about 1% to about 160%, about1% to about 140%, about 1% to about 120%, about 1% to about 100%, about1% to about 90%, about 1% to about 80%, about 1% to about 70%, about 1%to about 60%, about 1% to about 50%, about 1% to about 45%, about 1% toabout 40%, about 1% to about 35%, about 1% to about 30%, about 1% toabout 25%, about 1% to about 20%, about 1% to about 15%, about 1% toabout 10%, about 1% to about 5%, about 5% to about 800%, about 5% toabout 750%, about 5% to about 700%, about 5% to about 650%, about 5% toabout 600%, about 5% to about 550%, about 5% to about 500%, about 5% toabout 450%, about 5% to about 400%, about 5% to about 350%, about 5% toabout 300%, about 5% to about 280%, about 5% to about 260%, about 5% toabout 240%, about 5% to about 220%, about 5% to about 200%, about 5% toabout 180%, about 5% to about 160%, about 5% to about 140%, about 5% toabout 120%, about 5% to about 100%, about 5% to about 90%, about 5% toabout 80%, about 5% to about 70%, about 5% to about 60%, about 5% toabout 50%, about 5% to about 45%, about 5% to about 40%, about 5% toabout 35%, about 5% to about 30%, about 5% to about 25%, about 5% toabout 20%, about 5% to about 15%, about 5% to about 10%, about 10% toabout 800%, about 10% to about 750%, about 10% to about 700%, about 10%to about 650%, about 10% to about 600%, about 10% to about 550%, about10% to about 500%, about 10% to about 450%, about 10% to about 400%,about 10% to about 350%, about 10% to about 300%, about 10% to about280%, about 10% to about 260%, about 10% to about 240%, about 10% toabout 220%, about 10% to about 200%, about 10% to about 180%, about 10%to about 160%, about 10% to about 140%, about 10% to about 120%, about10% to about 100%, about 10% to about 90%, about 10% to about 80%, about10% to about 70%, about 10% to about 60%, about 10% to about 50%, about10% to about 45%, about 10% to about 40%, about 10% to about 35%, about10% to about 30%, about 10% to about 25%, about 10% to about 20%, about10% to about 15%, about 15% to about 800%, about 15% to about 750%,about 15% to about 700%, about 15% to about 650%, about 15% to about600%, about 15% to about 550%, about 15% to about 500%, about 15% toabout 450%, about 15% to about 400%, about 15% to about 350%, about 15%to about 300%, about 15% to about 280%, about 15% to about 260%, about15% to about 240%, about 15% to about 220%, about 15% to about 200%,about 15% to about 180%, about 15% to about 160%, about 15% to about140%, about 15% to about 120%, about 15% to about 100%, about 15% toabout 90%, about 15% to about 80%, about 15% to about 70%, about 15% toabout 60%, about 15% to about 50%, about 15% to about 45%, about 15% toabout 40%, about 15% to about 35%, about 15% to about 30%, about 15% toabout 25%, about 15% to about 20%, about 20% to about 800%, about 20% toabout 750%, about 20% to about 700%, about 20% to about 650%, about 20%to about 600%, about 20% to about 550%, about 20% to about 500%, about20% to about 450%, about 20% to about 400%, about 20% to about 350%,about 20% to about 300%, about 20% to about 280%, about 20% to about260%, about 20% to about 240%, about 20% to about 220%, about 20% toabout 200%, about 20% to about 180%, about 20% to about 160%, about 20%to about 140%, about 20% to about 120%, about 20% to about 100%, about20% to about 90%, about 20% to about 80%, about 20% to about 70%, about20% to about 60%, about 20% to about 50%, about 20% to about 45%, about20% to about 40%, about 20% to about 35%, about 20% to about 30%, about20% to about 25%, about 25% to about 800%, about 25% to about 750%,about 25% to about 700%, about 25% to about 650%, about 25% to about600%, about 25% to about 550%, about 25% to about 500%, about 25% toabout 450%, about 25% to about 400%, about 25% to about 350%, about 25%to about 300%, about 25% to about 280%, about 25% to about 260%, about25% to about 240%, about 25% to about 220%, about 25% to about 200%,about 25% to about 180%, about 25% to about 160%, about 25% to about140%, about 25% to about 120%, about 25% to about 100%, about 25% toabout 90%, about 25% to about 80%, about 25% to about 70%, about 25% toabout 60%, about 25% to about 50%, about 25% to about 45%, about 25% toabout 40%, about 25% to about 35%, about 35% to about 800%, about 35% toabout 750%, about 35% to about 700%, about 35% to about 650%, about 35%to about 600%, about 35% to about 550%, about 35% to about 500%, about35% to about 450%, about 35% to about 400%, about 35% to about 350%,about 35% to about 300%, about 35% to about 280%, about 35% to about260%, about 35% to about 240%, about 35% to about 220%, about 35% toabout 200%, about 35% to about 180%, about 35% to about 160%, about 35%to about 140%, about 35% to about 120%, about 35% to about 100%, about35% to about 90%, about 35% to about 80%, about 35% to about 70%, about35% to about 60%, about 35% to about 50%, about 35% to about 45%, about35% to about 40%, about 40% to about 800%, about 40% to about 750%,about 40% to about 700%, about 40% to about 650%, about 40% to about600%, about 40% to about 550%, about 40% to about 500%, about 40% toabout 450%, about 40% to about 400%, about 40% to about 350%, about 40%to about 300%, about 40% to about 280%, about 40% to about 260%, about40% to about 240%, about 40% to about 220%, about 40% to about 200%,about 40% to about 180%, about 40% to about 160%, about 40% to about140%, about 40% to about 120%, about 40% to about 100%, about 40% toabout 90%, about 40% to about 80%, about 40% to about 70%, about 40% toabout 60%, about 40% to about 50%, about 40% to about 45%, about 45% toabout 800%, about 45% to about 750%, about 45% to about 700%, about 45%to about 650%, about 45% to about 600%, about 45% to about 550%, about45% to about 500%, about 45% to about 450%, about 45% to about 400%,about 45% to about 350%, about 45% to about 300%, about 45% to about280%, about 45% to about 260%, about 45% to about 240%, about 45% toabout 220%, about 45% to about 200%, about 45% to about 180%, about 45%to about 160%, about 45% to about 140%, about 45% to about 120%, about45% to about 100%, about 45% to about 90%, about 45% to about 80%, about45% to about 70%, about 45% to about 60%, about 45% to about 50%, about50% to about 800%, about 50% to about 750%, about 50% to about 700%,about 50% to about 650%, about 50% to about 600%, about 50% to about550%, about 50% to about 500%, about 50% to about 450%, about 50% toabout 400%, about 50% to about 350%, about 50% to about 300%, about 50%to about 280%, about 50% to about 260%, about 50% to about 240%, about50% to about 220%, about 50% to about 200%, about 50% to about 180%,about 50% to about 160%, about 50% to about 140%, about 50% to about120%, about 50% to about 100%, about 50% to about 90%, about 50% toabout 80%, about 50% to about 70%, about 50% to about 60%, about 60% toabout 800%, about 60% to about 750%, about 60% to about 700%, about 60%to about 650%, about 60% to about 600%, about 60% to about 550%, about60% to about 500%, about 60% to about 450%, about 60% to about 400%,about 60% to about 350%, about 60% to about 300%, about 60% to about280%, about 60% to about 260%, about 60% to about 240%, about 60% toabout 220%, about 60% to about 200%, about 60% to about 180%, about 60%to about 160%, about 60% to about 140%, about 60% to about 120%, about60% to about 100%, about 60% to about 90%, about 60% to about 80%, about60% to about 70%, about 70% to about 800%, about 70% to about 750%,about 70% to about 700%, about 70% to about 650%, about 70% to about600%, about 70% to about 550%, about 70% to about 500%, about 70% toabout 450%, about 70% to about 400%, about 70% to about 350%, about 70%to about 300%, about 70% to about 280%, about 70% to about 260%, about70% to about 240%, about 70% to about 220%, about 70% to about 200%,about 70% to about 180%, about 70% to about 160%, about 70% to about140%, about 70% to about 120%, about 70% to about 100%, about 70% toabout 90%, about 70% to about 80%, about 80% to about 800%, about 80% toabout 750%, about 80% to about 700%, about 80% to about 650%, about 80%to about 600%, about 80% to about 550%, about 80% to about 500%, about80% to about 450%, about 80% to about 400%, about 80% to about 350%,about 80% to about 300%, about 80% to about 280%, about 80% to about260%, about 80% to about 240%, about 80% to about 220%, about 80% toabout 200%, about 80% to about 180%, about 80% to about 160%, about 80%to about 140%, about 80% to about 120%, about 80% to about 100%, about80% to about 90%, about 90% to about 800%, about 90% to about 750%,about 90% to about 700%, about 90% to about 650%, about 90% to about600%, about 90% to about 550%, about 90% to about 500%, about 90% toabout 450%, about 90% to about 400%, about 90% to about 350%, about 90%to about 300%, about 90% to about 280%, about 90% to about 260%, about90% to about 240%, about 90% to about 220%, about 90% to about 200%,about 90% to about 180%, about 90% to about 160%, about 90% to about140%, about 90% to about 120%, about 90% to about 100%, about 100% toabout 800%, about 100% to about 750%, about 100% to about 700%, about100% to about 650%, about 100% to about 600%, about 100% to about 550%,about 100% to about 500%, about 100% to about 450%, about 100% to about400%, about 100% to about 350%, about 100% to about 300%, about 100% toabout 280%, about 100% to about 260%, about 100% to about 240%, about100% to about 220%, about 100% to about 200%, about 100% to about 180%,about 100% to about 160%, about 100% to about 140%, about 100% to about120%, about 120% to about 800%, about 120% to about 750%, about 120% toabout 700%, about 120% to about 650%, about 120% to about 600%, about120% to about 550%, about 120% to about 500%, about 120% to about 450%,about 120% to about 400%, about 120% to about 350%, about 120% to about300%, about 120% to about 280%, about 120% to about 260%, about 120% toabout 240%, about 120% to about 220%, about 120% to about 200%, about120% to about 180%, about 120% to about 160%, about 120% to about 140%,about 140% to about 800%, about 140% to about 750%, about 140% to about700%, about 140% to about 650%, about 140% to about 600%, about 140% toabout 550%, about 140% to about 500%, about 140% to about 450%, about140% to about 400%, about 140% to about 350%, about 140% to about 300%,about 140% to about 280%, about 140% to about 260%, about 140% to about240%, about 140% to about 220%, about 140% to about 200%, about 140% toabout 180%, about 140% to about 160%, about 160% to about 800%, about160% to about 750%, about 160% to about 700%, about 160% to about 650%,about 160% to about 600%, about 160% to about 550%, about 160% to about500%, about 160% to about 450%, about 160% to about 400%, about 160% toabout 350%, about 160% to about 300%, about 160% to about 280%, about160% to about 260%, about 160% to about 240%, about 160% to about 220%,about 160% to about 200%, about 160% to about 180%, about 180% to about800%, about 180% to about 750%, about 180% to about 700%, about 180% toabout 650%, about 180% to about 600%, about 180% to about 550%, about180% to about 500%, about 180% to about 450%, about 180% to about 400%,about 180% to about 350%, about 180% to about 300%, about 180% to about280%, about 180% to about 260%, about 180% to about 240%, about 180% toabout 220%, about 180% to about 200%, about 200% to about 800%, about200% to about 750%, about 200% to about 700%, about 200% to about 650%,about 200% to about 600%, about 200% to about 550%, about 200% to about500%, about 200% to about 450%, about 200% to about 400%, about 200% toabout 350%, about 200% to about 300%, about 200% to about 280%, about200% to about 260%, about 200% to about 240%, about 200% to about 220%,about 220% to about 800%, about 220% to about 750%, about 220% to about700%, about 220% to about 650%, about 220% to about 600%, about 220% toabout 550%, about 220% to about 500%, about 220% to about 450%, about220% to about 400%, about 220% to about 350%, about 220% to about 300%,about 220% to about 280%, about 220% to about 260%, about 220% to about240%, about 240% to about 800%, about 240% to about 750%, about 240% toabout 700%, about 240% to about 650%, about 240% to about 600%, about240% to about 550%, about 240% to about 500%, about 240% to about 450%,about 240% to about 400%, about 240% to about 350%, about 240% to about300%, about 240% to about 280%, about 240% to about 260%, about 260% toabout 800%, about 260% to about 750%, about 260% to about 700%, about260% to about 650%, about 260% to about 600%, about 260% to about 550%,about 260% to about 500%, about 260% to about 450%, about 260% to about400%, about 260% to about 350%, about 260% to about 300%, about 260% toabout 280%, about 280% to about 800%, about 280% to about 750%, about280% to about 700%, about 280% to about 650%, about 280% to about 600%,about 280% to about 550%, about 280% to about 500%, about 280% to about450%, about 280% to about 400%, about 280% to about 350%, about 280% toabout 300%, about 300% to about 800%, about 300% to about 750%, about300% to about 700%, about 300% to about 650%, about 300% to about 600%,about 300% to about 550%, about 300% to about 500%, about 300% to about450%, about 300% to about 400%, about 300% to about 350%, about 350% toabout 800%, about 350% to about 750%, about 350% to about 700%, about350% to about 650%, about 350% to about 600%, about 350% to about 550%,about 350% to about 500%, about 350% to about 450%, about 350% to about400%, about 400% to about 800%, about 400% to about 750%, about 400% toabout 700%, about 400% to about 650%, about 400% to about 600%, about400% to about 550%, about 400% to about 500%, about 400% to about 450%,about 450% to about 800%, about 450% to about 750%, about 450% to about700%, about 450% to about 650%, about 450% to about 600%, about 450% toabout 550%, about 450% to about 500%, about 500% to about 800%, about500% to about 750%, about 500% to about 700%, about 500% to about 650%,about 500% to about 600%, about 500% to about 550%, about 550% to about800%, about 550% to about 750%, about 550% to about 700%, about 550% toabout 650%, about 550% to about 600%, about 600% to about 800%, about600% to about 750%, about 600% to about 700%, about 600% to about 650%,about 650% to about 800%, about 650% to about 750%, about 650% to about700%, about 700% to about 800%, about 700% to about 750%, or about 750%to about 800%) of one or more of any of the cytokines or chemokines orcytotoxicity granules or regulatory molecules described herein (e.g.,one or more of any of IL-2, IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9,IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33, leukotriene B4,CCL5, TNFα, granzymes, perforin, TGFβ, STAT3, STAT4, STAT5, RORγT,FOXP3, and GATA3) (e.g., as compared to the level of the one or morecytokines, chemokines, cytotoxicity granules, and regulatory moleculesin a control not contacted with any of the multi-chain chimericpolypeptides described herein).

Methods of Inducing or Increasing Proliferation of an Immune Cell

Also provided herein are methods of inducing or increasing proliferationof an immune cell (e.g., any of the exemplary immune cells describedherein or known in the art) that include contacting an immune cell withan effective amount of any of the multi-chain chimeric polypeptidesdescribed herein or any of the compositions (e.g., pharmaceuticalcompositions) described herein. In some examples, the immune cell iscontacted in vitro (e.g., in a suitable liquid culture medium underconditions sufficient to result in stimulation of the immune cell).

In some examples, the immune cell has been previously obtained from asubject (e.g., a mammal, e.g., a human). Some embodiments of thesemethods further include obtaining the immune cell from the subject priorto the contacting step.

In some examples, the immune cell is contacted in vivo. In suchembodiments, the multi-chain chimeric polypeptide is administered to asubject (e.g., a mammal, e.g., a human) in an amount sufficient toresult in stimulation of an immune cell in the subject.

In some examples of any of the methods described herein, the immune cellcan be an immature thymocyte, a peripheral blood lymphocyte, a naïve Tcell, a pluripotent Th cell precursor, a lymphoid progenitor cell, aTreg cell, a memory T cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ T cell, atumor-infiltrating T cell, a CD8⁺ T cell, a CD4⁺ T cell, a naturalkiller T cell, a mast cell, a macrophage, a neutrophil, a dendriticcell, a basophil, an eosinophil, or a natural killer cell, or acombination thereof.

In some examples, the immune cell has previously beengenetically-modified to express a chimeric antigen receptor or arecombinant T-cell receptor. In some examples, the immune cell (e.g.,any of the immune cells described herein) has previously beengenetically-modified to express a co-stimulatory molecule (e.g., CD28).

Some embodiments of these methods can further include, after thecontacting step, introducing into the immune cell (e.g., any of theimmune cells described herein) a nucleic acid encoding a chimericantigen-receptor or a recombinant T-cell receptor. Some embodiments ofthese methods can further include, after the contacting step,introducing into the immune cell (e.g., any of the immune cellsdescribed herein) a nucleic acid encoding a co-stimulatory molecule(e.g., CD28).

Some embodiments of these methods can further include administering atherapeutically effective amount of the immune cell to a subject in needthereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosedas having an age-related disease or condition. Non-limiting examples ofage-related diseases or disorders include: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identifiedor diagnosed as having a cancer. Non-limiting examples of cancersinclude: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosedor identified as having an infectious disease. Non-limiting examples ofinfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, or influenza virus.

Detection of the proliferation of an immune cell can be performed usingmethods known in the art, e.g., cytometry (e.g., fluorescence-assistedflow cytometry), microscopy, and immunofluorescence microscopy, e.g., bycomparing the rate of increase in the concentration of the immune cellin a sample not contacted with a multi-chain chimeric polypeptide to therate of increase in the concentration of the immune cell in a similarsample contacted with any of the multi-chain chimeric polypeptidesdescribed herein).

In other examples, the proliferation of an immune cell can be indirectlydetected by detecting an increase in the level of one or more cytokinesor chemokines or cytotoxicity granules or regulatory molecules secretedor upregulated by proliferating immune cells (e.g., one or more of IL-2,IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15,IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFα, granzymes,perforin, TGFβ, STAT3, STAT4, STAT5, RORγT, FOXP3, and GATA3) (e.g., ascompared to the level of the one or more cytokines, chemokines,cytotoxicity granules, and regulatory molecules in a control notcontacted with any of the multi-chain chimeric polypeptides describedherein).

In some embodiments, the methods provided herein can result in anincrease (e.g., about 1% to about 800% increase, or any of the subrangesof this range described herein) in the rate of increase in theconcentration of the immune cell in a sample contacted with any of themulti-chain chimeric polypeptides described herein as compared to therate of increase in a similar control sample not contacted with any ofthe multi-chain chimeric polypeptides described herein.

Methods of Inducing Differentiation of an Immune Cell

Also provided herein are method of inducing differentiation of an immunecell (e.g., any of the exemplary immune cells described herein or knownin the art) into a memory or memory-like immune cell that includecontacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides described herein or any of thecompositions (e.g., pharmaceutical compositions) described herein. Insome examples, the immune cell is contacted in vitro (e.g., in asuitable liquid culture medium under conditions sufficient to result instimulation of the immune cell).

In some examples, the immune cell has been previously obtained from asubject (e.g., a mammal, e.g., a human). Some embodiments of thesemethods further include obtaining the immune cell from the subject priorto the contacting step.

In some examples, the immune cell is contacted in vivo. In suchembodiments, the multi-chain chimeric polypeptide is administered to asubject (e.g., a mammal, e.g., a human) in an amount sufficient toresult in stimulation of an immune cell in the subject.

In some examples of any of the methods described herein, the immune cellcan be an immature thymocyte, a peripheral blood lymphocyte, a naïve Tcell, a pluripotent Th cell precursor, a lymphoid progenitor cell, aTreg cell, a Th17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell,a Th3 cell, γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+T cell, a CD4+ T cell, a natural killer T cell, a mast cell, amacrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil,or a natural killer cell, or a combination thereof.

In some examples, the immune cell has previously beengenetically-modified to express a chimeric antigen receptor or arecombinant T-cell receptor. In some examples, the immune cell (e.g.,any of the immune cells described herein) has previously beengenetically-modified to express a co-stimulatory molecule (e.g., CD28).

In some examples, an effective amount of any of the multi-chain chimericpolypeptides described herein or any of the compositions (e.g.,pharmaceutical compositions) described herein is combined with ananti-TF IgG1 antibody to create a memory or memory like immune cell.

Some embodiments of these methods can further include, after thecontacting step, introducing into the immune cell (e.g., any of theimmune cells described herein) a nucleic acid encoding a chimericantigen-receptor or a recombinant T-cell receptor. Some embodiments ofthese methods can further include, after the contacting step,introducing into the immune cell (e.g., any of the immune cellsdescribed herein) a nucleic acid encoding a co-stimulatory molecule(e.g., CD28).

Some embodiments of these methods can further include administering atherapeutically effective amount of the immune cell to a subject in needthereof (e.g., any of the exemplary subjects described herein).

In some examples, the subject can be a subject identified or diagnosedas having an age-related disease or condition. Non-limiting examples ofage-related diseases or disorders include: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

In some examples, the subject can be a subject that has been identifiedor diagnosed as having a cancer. Non-limiting examples of cancersinclude: solid tumor, hematological tumor, sarcoma, osteosarcoma,glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma,osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma,B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocyticleukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia(CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes(MDS), cutaneous T-cell lymphoma, retinoblastoma, stomach cancer,urothelial carcinoma, lung cancer, renal cell carcinoma, gastric andesophageal cancer, pancreatic cancer, prostate cancer, breast cancer,colorectal cancer, ovarian cancer, non-small cell lung carcinoma,squamous cell head and neck carcinoma, endometrial cancer, cervicalcancer, liver cancer, and hepatocellular carcinoma.

In some examples, the subject can be a subject that has been diagnosedor identified as having an infectious disease. Non-limiting examples ofinfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, or influenza virus. In someexamples, the immune cell is a NK cell, and the detection of a memory NKcell can include, e.g., the detection of the level of one or more ofIL-12, IL-18, IL-33, CD25, CD69, CD62L, STAT4, Zbtb32, DNAM-1, NKp30,NKp44, NKp46, BIM, Noxa, SOCS1, BNIP3, BNIP3L, IFN-γ, CXCL16, CXCR6,NKG2D, TRAIL, CD49, Ly49D, CD49b, and Ly79H. A description of NK memorycells and methods of detecting the same is described in O'Sullivan etal., Immunity 43:634-645, 2015.

In some examples, the immune cell is a T cell, and the detection ofmemory T cells can include, e.g., the detection of the level ofexpression of one or more of CD45RO, CCR7, L-selectin (CD62L), CD44,CD45RA, integrin αeβ7, CD43, CD27, CD28, IL-7Rα, CD95, CXCR3, and LFA-1.In some examples, the immune cell is a B cell and the detection ofmemory B cells can include, e.g., the detection of the level ofexpression of CD27. Other types and markers of memory or memory-likeimmune cells are known in the art.

Methods of Treatment

Also provided herein are methods of treating a subject in need thereof(e.g., any of the exemplary subjects described herein or known in theart) that include administering to the subject a therapeuticallyeffective amount of any of the multi-chain chimeric polypeptidesdescribed herein or any of the compositions (e.g., pharmaceuticalcompositions) described herein.

In some embodiments of these methods, the subject has been identified ordiagnosed as having a cancer. Non-limiting examples of cancer include:solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma,neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma,B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cellnon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia(CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML),acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS),cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelialcarcinoma, lung cancer, renal cell carcinoma, gastric and esophagealcancer, pancreatic cancer, prostate cancer, breast cancer, colorectalcancer, ovarian cancer, non-small cell lung carcinoma, squamous cellhead and neck carcinoma, endometrial cancer, cervical cancer, livercancer, and hepatocellular carcinoma. In some embodiments, these methodscan result in a reduction in the number, severity, or frequency of oneor more symptoms of the cancer in the subject (e.g., as compared to thenumber, severity, or frequency of the one or more symptoms of the cancerin the subject prior to treatment). In some embodiments, these methodscan result in a reduction (e.g., about 1% reduction to about 99%reduction, about 1% reduction to about 95% reduction, about 1% reductionto about 90% reduction, about 1% reduction to about 85% reduction, about1% reduction to about 80% reduction, about 1% reduction to about 75%reduction, about 1% reduction to about 70% reduction, about 1% reductionto about 65% reduction, about 1% reduction to about 60% reduction, about1% reduction to about 55% reduction, about 1% reduction to about 50%reduction, about 1% reduction to about 45% reduction, about 1% reductionto about 40% reduction, about 1% reduction to about 35% reduction, about1% reduction to about 30% reduction, about 1% reduction to about 25%reduction, about 1% reduction to about 20% reduction, about 1% reductionto about 15% reduction, about 1% reduction to about 10% reduction, about1% reduction to about 5% reduction, about 5% reduction to about 99%reduction, about 5% reduction to about 95% reduction, about 5% reductionto about 90% reduction, about 5% reduction to about 85% reduction, about5% reduction to about 80% reduction, about 5% reduction to about 75%reduction, about 5% reduction to about 70% reduction, about 5% reductionto about 65% reduction, about 5% reduction to about 60% reduction, about5% reduction to about 55% reduction, about 5% reduction to about 50%reduction, about 5% reduction to about 45% reduction, about 5% reductionto about 40% reduction, about 5% reduction to about 35% reduction, about5% reduction to about 30% reduction, about 5% reduction to about 25%reduction, about 5% reduction to about 20% reduction, about 5% reductionto about 15% reduction, about 5% reduction to about 10% reduction, about10% reduction to about 99% reduction, about 10% reduction to about 95%reduction, about 10% reduction to about 90% reduction, about 10%reduction to about 85% reduction, about 10% reduction to about 80%reduction, about 10% reduction to about 75% reduction, about 10%reduction to about 70% reduction, about 10% reduction to about 65%reduction, about 10% reduction to about 60% reduction, about 10%reduction to about 55% reduction, about 10% reduction to about 50%reduction, about 10% reduction to about 45% reduction, about 10%reduction to about 40% reduction, about 10% reduction to about 35%reduction, about 10% reduction to about 30% reduction, about 10%reduction to about 25% reduction, about 10% reduction to about 20%reduction, about 10% reduction to about 15% reduction, about 15%reduction to about 99% reduction, about 15% reduction to about 95%reduction, about 15% reduction to about 90% reduction, about 15%reduction to about 85% reduction, about 15% reduction to about 80%reduction, about 15% reduction to about 75% reduction, about 15%reduction to about 70% reduction, about 15% reduction to about 65%reduction, about 15% reduction to about 60% reduction, about 15%reduction to about 55% reduction, about 15% reduction to about 50%reduction, about 15% reduction to about 45% reduction, about 15%reduction to about 40% reduction, about 15% reduction to about 35%reduction, about 15% reduction to about 30% reduction, about 15%reduction to about 25% reduction, about 15% reduction to about 20%reduction, about 20% reduction to about 99% reduction, about 20%reduction to about 95% reduction, about 20% reduction to about 90%reduction, about 20% reduction to about 85% reduction, about 20%reduction to about 80% reduction, about 20% reduction to about 75%reduction, about 20% reduction to about 70% reduction, about 20%reduction to about 65% reduction, about 20% reduction to about 60%reduction, about 20% reduction to about 55% reduction, about 20%reduction to about 50% reduction, about 20% reduction to about 45%reduction, about 20% reduction to about 40% reduction, about 20%reduction to about 35% reduction, about 20% reduction to about 30%reduction, about 20% reduction to about 25% reduction, about 25%reduction to about 99% reduction, about 25% reduction to about 95%reduction, about 25% reduction to about 90% reduction, about 25%reduction to about 85% reduction, about 25% reduction to about 80%reduction, about 25% reduction to about 75% reduction, about 25%reduction to about 70% reduction, about 25% reduction to about 65%reduction, about 25% reduction to about 60% reduction, about 25%reduction to about 55% reduction, about 25% reduction to about 50%reduction, about 25% reduction to about 45% reduction, about 25%reduction to about 40% reduction, about 25% reduction to about 35%reduction, about 25% reduction to about 30% reduction, about 30%reduction to about 99% reduction, about 30% reduction to about 95%reduction, about 30% reduction to about 90% reduction, about 30%reduction to about 85% reduction, about 30% reduction to about 80%reduction, about 30% reduction to about 75% reduction, about 30%reduction to about 70% reduction, about 30% reduction to about 65%reduction, about 30% reduction to about 60% reduction, about 30%reduction to about 55% reduction, about 30% reduction to about 50%reduction, about 30% reduction to about 45% reduction, about 30%reduction to about 40% reduction, about 30% reduction to about 35%reduction, about 35% reduction to about 99% reduction, about 35%reduction to about 95% reduction, about 35% reduction to about 90%reduction, about 35% reduction to about 85% reduction, about 35%reduction to about 80% reduction, about 35% reduction to about 75%reduction, about 35% reduction to about 70% reduction, about 35%reduction to about 65% reduction, about 35% reduction to about 60%reduction, about 35% reduction to about 55% reduction, about 35%reduction to about 50% reduction, about 35% reduction to about 45%reduction, about 35% reduction to about 40% reduction, about 40%reduction to about 99% reduction, about 40% reduction to about 95%reduction, about 40% reduction to about 90% reduction, about 40%reduction to about 85% reduction, about 40% reduction to about 80%reduction, about 40% reduction to about 75% reduction, about 40%reduction to about 70% reduction, about 40% reduction to about 65%reduction, about 40% reduction to about 60% reduction, about 40%reduction to about 55% reduction, about 40% reduction to about 50%reduction, about 40% reduction to about 45% reduction, about 45%reduction to about 99% reduction, about 45% reduction to about 95%reduction, about 45% reduction to about 90% reduction, about 45%reduction to about 85% reduction, about 45% reduction to about 80%reduction, about 45% reduction to about 75% reduction, about 45%reduction to about 70% reduction, about 45% reduction to about 65%reduction, about 45% reduction to about 60% reduction, about 45%reduction to about 55% reduction, about 45% reduction to about 50%reduction, about 50% reduction to about 99% reduction, about 50%reduction to about 95% reduction, about 50% reduction to about 90%reduction, about 50% reduction to about 85% reduction, about 50%reduction to about 80% reduction, about 50% reduction to about 75%reduction, about 50% reduction to about 70% reduction, about 50%reduction to about 65% reduction, about 50% reduction to about 60%reduction, about 50% reduction to about 55% reduction, about 55%reduction to about 99% reduction, about 55% reduction to about 95%reduction, about 55% reduction to about 90% reduction, about 55%reduction to about 85% reduction, about 55% reduction to about 80%reduction, about 55% reduction to about 75% reduction, about 55%reduction to about 70% reduction, about 55% reduction to about 65%reduction, about 55% reduction to about 60% reduction, about 60%reduction to about 99% reduction, about 60% reduction to about 95%reduction, about 60% reduction to about 90% reduction, about 60%reduction to about 85% reduction, about 60% reduction to about 80%reduction, about 60% reduction to about 75% reduction, about 60%reduction to about 70% reduction, about 60% reduction to about 65%reduction, about 65% reduction to about 99% reduction, about 65%reduction to about 95% reduction, about 65% reduction to about 90%reduction, about 65% reduction to about 85% reduction, about 65%reduction to about 80% reduction, about 65% reduction to about 75%reduction, about 65% reduction to about 70% reduction, about 70%reduction to about 99% reduction, about 70% reduction to about 95%reduction, about 70% reduction to about 90% reduction, about 70%reduction to about 85% reduction, about 70% reduction to about 80%reduction, about 70% reduction to about 75% reduction, about 75%reduction to about 99% reduction, about 75% reduction to about 95%reduction, about 75% reduction to about 90% reduction, about 75%reduction to about 85% reduction, about 75% reduction to about 80%reduction, about 80% reduction to about 99% reduction, about 80%reduction to about 95% reduction, about 80% reduction to about 90%reduction, about 80% reduction to about 85% reduction, about 85%reduction to about 99% reduction, about 85% reduction to about 95%reduction, about 85% reduction to about 90% reduction, about 90%reduction to about 99% reduction, about 90% reduction to about 95%reduction, or about 95% reduction to about 99% reduction) in the volumeof one or more solid tumors in the subject (e.g., as compared to thevolume of the one or more solid tumors prior to treatment or at thestart of treatment). In some embodiments, the these methods can reduce(e.g., about 1% reduction to about 99% reduction, or any of thesubranges of this range described herein) the risk of developing ametastasis or developing one or more additional metastasis in a subject(e.g., as compared to the risk of developing a metastasis or developingone or more additional metastasis in a subject prior to treatment or ina similar subject or a population of subjects administered a differenttreatment).

In some examples of these methods, the subject has been identified ordiagnosed as having an aging-related disease or condition. Non-limitingexamples of aging-related diseases and conditions include Alzheimer'sdisease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction. In some examples, thesemethods can result in a reduction in the number, severity, or frequencyof one or more symptoms of the aging-related disease or condition in thesubject (e.g., as compared to the number, severity, or frequency of theone or more symptoms of the aging-related disease or condition in thesubject prior to treatment). In some examples, the methods can result ina decrease (e.g., about 1% decrease to about 99% decrease, an about 1%decrease to about 95% decrease, about 1% decrease to about 90% decrease,about 1% decrease to about 85% decrease, about 1% decrease to about 80%decrease, about 1% decrease to about 75% decrease, about 1% to about 70%decrease, about 1% decrease to about 65% decrease, about 1% decrease toabout 60% decrease, about 1% decrease to about 55% decrease, about 1%decrease to about 50% decrease, about 1% decrease to about 45% decrease,about 1% decrease to about 40% decrease, about 1% decrease to about 35%decrease, about 1% decrease to about 30% decrease, about 1% decrease toabout 25% decrease, about 1% decrease to about 20% decrease, about 1%decrease to about 15% decrease, about 1% decrease to about 10% decrease,about 1% decrease to about 5% decrease, about 5% decrease to about 99%decrease, an about 5% decrease to about 95% decrease, about 5% decreaseto about 90% decrease, about 5% decrease to about 85% decrease, about 5%decrease to about 80% decrease, about 5% decrease to about 75% decrease,about 5% to about 70% decrease, about 5% decrease to about 65% decrease,about 5% decrease to about 60% decrease, about 5% decrease to about 55%decrease, about 5% decrease to about 50% decrease, about 5% decrease toabout 45% decrease, about 5% decrease to about 40% decrease, about 5%decrease to about 35% decrease, about 5% decrease to about 30% decrease,about 5% decrease to about 25% decrease, about 5% decrease to about 20%decrease, about 5% decrease to about 15% decrease, about 5% decrease toabout 10% decrease, about 10% decrease to about 99% decrease, an about10% decrease to about 95% decrease, about 10% decrease to about 90%decrease, about 10% decrease to about 85% decrease, about 10% decreaseto about 80% decrease, about 10% decrease to about 75% decrease, about10% to about 70% decrease, about 10% decrease to about 65% decrease,about 10% decrease to about 60% decrease, about 10% decrease to about55% decrease, about 10% decrease to about 50% decrease, about 10%decrease to about 45% decrease, about 10% decrease to about 40%decrease, about 10% decrease to about 35% decrease, about 10% decreaseto about 30% decrease, about 10% decrease to about 25% decrease, about10% decrease to about 20% decrease, about 10% decrease to about 15%decrease, about 15% decrease to about 99% decrease, an about 15%decrease to about 95% decrease, about 15% decrease to about 90%decrease, about 15% decrease to about 85% decrease, about 15% decreaseto about 80% decrease, about 15% decrease to about 75% decrease, about15% to about 70% decrease, about 15% decrease to about 65% decrease,about 15% decrease to about 60% decrease, about 15% decrease to about55% decrease, about 15% decrease to about 50% decrease, about 15%decrease to about 45% decrease, about 15% decrease to about 40%decrease, about 15% decrease to about 35% decrease, about 15% decreaseto about 30% decrease, about 15% decrease to about 25% decrease, about15% decrease to about 20% decrease, about 20% decrease to about 99%decrease, an about 20% decrease to about 95% decrease, about 20%decrease to about 90% decrease, about 20% decrease to about 85%decrease, about 20% decrease to about 80% decrease, about 20% decreaseto about 75% decrease, about 20% to about 70% decrease, about 20%decrease to about 65% decrease, about 20% decrease to about 60%decrease, about 20% decrease to about 55% decrease, about 20% decreaseto about 50% decrease, about 20% decrease to about 45% decrease, about20% decrease to about 40% decrease, about 20% decrease to about 35%decrease, about 20% decrease to about 30% decrease, about 20% decreaseto about 25% decrease, about 25% decrease to about 99% decrease, anabout 25% decrease to about 95% decrease, about 25% decrease to about90% decrease, about 25% decrease to about 85% decrease, about 25%decrease to about 80% decrease, about 25% decrease to about 75%decrease, about 25% to about 70% decrease, about 25% decrease to about65% decrease, about 25% decrease to about 60% decrease, about 25%decrease to about 55% decrease, about 25% decrease to about 50%decrease, about 25% decrease to about 45% decrease, about 25% decreaseto about 40% decrease, about 25% decrease to about 35% decrease, about25% decrease to about 30% decrease, about 30% decrease to about 99%decrease, an about 30% decrease to about 95% decrease, about 30%decrease to about 90% decrease, about 30% decrease to about 85%decrease, about 30% decrease to about 80% decrease, about 30% decreaseto about 75% decrease, about 30% to about 70% decrease, about 30%decrease to about 65% decrease, about 30% decrease to about 60%decrease, about 30% decrease to about 55% decrease, about 30% decreaseto about 50% decrease, about 30% decrease to about 45% decrease, about30% decrease to about 40% decrease, about 30% decrease to about 35%decrease, about 35% decrease to about 99% decrease, an about 35%decrease to about 95% decrease, about 35% decrease to about 90%decrease, about 35% decrease to about 85% decrease, about 35% decreaseto about 80% decrease, about 35% decrease to about 75% decrease, about35% to about 70% decrease, about 35% decrease to about 65% decrease,about 35% decrease to about 60% decrease, about 35% decrease to about55% decrease, about 35% decrease to about 50% decrease, about 35%decrease to about 45% decrease, about 35% decrease to about 40%decrease, about 40% decrease to about 99% decrease, an about 40%decrease to about 95% decrease, about 40% decrease to about 90%decrease, about 40% decrease to about 85% decrease, about 40% decreaseto about 80% decrease, about 40% decrease to about 75% decrease, about40% to about 70% decrease, about 40% decrease to about 65% decrease,about 40% decrease to about 60% decrease, about 40% decrease to about55% decrease, about 40% decrease to about 50% decrease, about 40%decrease to about 45% decrease, about 45% decrease to about 99%decrease, an about 45% decrease to about 95% decrease, about 45%decrease to about 90% decrease, about 45% decrease to about 85%decrease, about 45% decrease to about 80% decrease, about 45% decreaseto about 75% decrease, about 45% to about 70% decrease, about 45%decrease to about 65% decrease, about 45% decrease to about 60%decrease, about 45% decrease to about 55% decrease, about 45% decreaseto about 50% decrease, about 50% decrease to about 99% decrease, anabout 50% decrease to about 95% decrease, about 50% decrease to about90% decrease, about 50% decrease to about 85% decrease, about 50%decrease to about 80% decrease, about 50% decrease to about 75%decrease, about 50% to about 70% decrease, about 50% decrease to about65% decrease, about 50% decrease to about 60% decrease, about 50%decrease to about 55% decrease, about 55% decrease to about 99%decrease, an about 55% decrease to about 95% decrease, about 55%decrease to about 90% decrease, about 55% decrease to about 85%decrease, about 55% decrease to about 80% decrease, about 55% decreaseto about 75% decrease, about 55% to about 70% decrease, about 55%decrease to about 65% decrease, about 55% decrease to about 60%decrease, about 60% decrease to about 99% decrease, an about 60%decrease to about 95% decrease, about 60% decrease to about 90%decrease, about 60% decrease to about 85% decrease, about 60% decreaseto about 80% decrease, about 60% decrease to about 75% decrease, about60% to about 70% decrease, about 60% decrease to about 65% decrease,about 65% decrease to about 99% decrease, an about 65% decrease to about95% decrease, about 65% decrease to about 90% decrease, about 65%decrease to about 85% decrease, about 65% decrease to about 80%decrease, about 65% decrease to about 75% decrease, about 65% to about70% decrease, about 70% decrease to about 99% decrease, an about 70%decrease to about 95% decrease, about 70% decrease to about 90%decrease, about 70% decrease to about 85% decrease, about 70% decreaseto about 80% decrease, about 70% decrease to about 75% decrease, about75% decrease to about 99% decrease, an about 75% decrease to about 95%decrease, about 75% decrease to about 90% decrease, about 75% decreaseto about 85% decrease, about 75% decrease to about 80% decrease, about80% decrease to about 99% decrease, an about 80% decrease to about 95%decrease, about 80% decrease to about 90% decrease, about 80% decreaseto about 85% decrease, about 85% decrease to about 99% decrease, anabout 85% decrease to about 95% decrease, about 85% decrease to about90% decrease, about 90% decrease to about 99% decrease, an about 90%decrease to about 95% decrease, or about 95% decrease to about 99%decrease) in the number of senescent cells in the subject (e.g., adecrease in the number of senescent cells in one or more specifictissues involved and/or implicated in the aging-related disease ordisorder in the subject), e.g., as compared to the number of senescentcells in the subject prior to treatment.

In some examples of these methods, the subject has been diagnosed oridentified as having an infectious disease. Non-limiting examples ofinfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, and influenza virus. In someembodiments, these methods can result in a decrease in the infectioustiter (e.g., viral titer) in a subject (e.g., as compared to theinfectious titer in the subject prior to treatment). In someembodiments, these methods can result in a reduction in the number,severity, or frequency of one or more symptoms of the infectious disease(e.g., viral infection) in the subject (e.g., as compared to the number,severity, or frequency of the one or more symptoms of the infectiousdisease in the subject prior to treatment).

The term “subject” refers to any mammal. In some embodiments, thesubject or “subject in need of treatment” may be a canine (e.g., a dog),feline (e.g., a cat), equine (e.g., a horse), ovine, bovine, porcine,caprine, primate, e.g., a simian (e.g., a monkey (e.g., marmoset,baboon), or an ape (e.g., a gorilla, chimpanzee, orangutan, or gibbon)or a human; or rodent (e.g., a mouse, a guinea pig, a hamster, or arat). In some embodiments, the subject or “subject in need of treatment”may be a non-human mammal, especially mammals that are conventionallyused as models for demonstrating therapeutic efficacy in humans (e.g.,murine, lapine, porcine, canine or primate animals) may be employed.

Methods of Killing a Cancer Cell, an Infected Cell, or a Senescent Cell

Also provided herein are methods of killing a cancer cell (e.g., any ofthe exemplary types of cancer described herein or known in the art), aninfected cell (e.g., a cell infected with any of the exemplary virusesdescribed herein or known in the art), or a senescent cell (e.g., asenescent cancer cell, a senescent fibroblast, or a senescentendothelial cell) in a subject in need thereof (e.g., any of theexemplary subjects described herein or known in the art) that includeadministering to the subject a therapeutically effective amount of anyof the multi-chain chimeric polypeptides described herein or any of thecompositions (e.g., pharmaceutical compositions) described herein.

In some embodiments of these methods, the subject has been identified ordiagnosed as having a cancer. Non-limiting examples of cancer include:solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma,neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma,B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cellnon-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia(CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML),acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS),cutaneous T-cell lymphoma, retinoblastoma, stomach cancer, urothelialcarcinoma, lung cancer, renal cell carcinoma, gastric and esophagealcancer, pancreatic cancer, prostate cancer, breast cancer, colorectalcancer, ovarian cancer, non-small cell lung carcinoma, squamous cellhead and neck carcinoma, endometrial cancer, cervical cancer, livercancer, and hepatocellular carcinoma.

In some examples of these methods, the subject has been identified ordiagnosed as having an aging-related disease or condition. Non-limitingexamples of aging-related diseases and conditions include Alzheimer'sdisease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

In some examples of these methods, the subject has been diagnosed oridentified as having an infectious disease. Non-limiting examples of aninfectious disease include infection with human immunodeficiency virus,cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus,smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus,and hepatitis C virus, papillomavirus, and influenza virus.

Senescent Cells

Senescence is a form of irreversible growth arrest accompanied byphenotypic changes, resistance to apoptosis and activation ofdamage-sensing signaling pathways. Cellular senescence was firstdescribed in cultured human fibroblast cells that lost their ability toproliferate, reaching permanent arrest after about 50 populationdoublings (referred to as the Hayflick limit). Senescence is considereda stress response that can be induced by a wide range of intrinsic andextrinsic insults, including oxidative and genotoxic stress, DNA damage,telomere attrition, oncogenic activation, mitochondrial dysfunction, orchemotherapeutic agents.

Senescent cells remain metabolically active and can influence the tissuehemostasis, disease and aging through their secretory phenotype.Senescence is considered as a physiologic process and is important inpromoting wound healing, tissue homeostasis, regeneration, and fibrosisregulation. For instance, transient induction of senescent cells isobserved during would healing and contributes to wound resolution.Perhaps one of the most important roles of senescence is its role intumor suppression. However, the accumulation of senescent cells alsodrives aging- and aging-related diseases and conditions. The senescentphenotype also can trigger chronic inflammatory responses andconsequently augment chronic inflammatory conditions to promote tumorgrowth. The connection between senescence and aging was initially basedon observations that senescent cells accumulate in aged tissue. The useof transgenic models has enabled the detection of senescent cellssystematically in many age-related pathologies. Strategies toselectively eliminate senescent cells has demonstrated that senescentcells can indeed play a causal role in aging and related pathologies.

Senescent cells display important and unique properties which includechanges in morphology, chromatin organization, gene expression, andmetabolism. There are several biochemical and functional propertiesassociated with cellular senescence, such as (i) increased expression ofp16 and p21, inhibitors of cyclin-dependent kinases, (ii) presence ofsenescence-associated β-galactosidase, a marker of lysosomal activity,(iii) appearance of senescence-associated heterochromatin foci anddownregulation of lamin B1 levels, (iv) resistance to apoptosis causedby an increased expression of anti-apoptotic BCL-family protein, and (v)upregulation of CD26 (DPP4), CD36 (Scavenger receptor), forkhead box 4(FOXO4), and secretory carrier membrane protein 4 (SCAMP4). Senescentcells also express an inflammatory signature, the so-calledsenescence-associated secretory phenotype (SASP). Through SASP, thesenescent cells produce a wide range of inflammatory cytokines (IL-6,IL-8), growth factors (TGF-β), chemokines (CCL-2), and matrixmetalloproteinases (MMP-3, MMP-9) that operate in a cell-autonomousmanner to reinforce senescence (autocrine effects) and communicate withand modify the microenvironment (paracrine effects). SASP factors cancontribute to tumor suppression by triggering senescence surveillance,an immune-mediated clearance of senescent cells. However, chronicinflammation is also a known driver of tumorigenesis, and accumulatingevidence indicates that chronic SASP can also boost cancer andaging-related diseases.

The secretion profile of senescent cells is context dependent. Forinstance, the mitochondrial dysfunction-associated senescence (MiDAS),induced by different mitochondrial dysfunction in human fibroblasts, ledto the appearance of a SASP that was deficient in IL-1-dependentinflammatory factors. A decrease in the NAD+/NADH ratio activated AMPKsignaling which induced MiDAS through the activation of p53. As aresult, p53 inhibited NF-κB signaling which is a crucial inducer ofpro-inflammatory SASP. In contrast, the cellular senescence caused bypersistent DNA damage in human cells induced an inflammatory SASP, whichwas dependent on the activation of ataxia-telangiectasia mutated (ATM)kinase but not on that of p53. In particular, the expression andsecretion levels of IL-6 and IL-8 were increased. It was alsodemonstrated that cellular senescence caused by the ectopic expressionp16INK4a and p21CIP1 induced the senescent phenotype in humanfibroblasts without an inflammatory SASP indicating that the growtharrest itself did not stimulate SASP.

One of the most defining characteristics of senescence is stable growtharrest. This is achieved by two important pathways, the p16/Rb and thep53/p21, both of which are central in tumor suppression. DNA damageresults in: (1) high deposition of γH2Ax (histone coding gene) and 53BP1(involved in DNA damage response) in chromatin: this leads to activationof a kinase cascade eventually resulting in p53 activation, and (2)activation of p16INK4a and ARF (both encoded by CDKN2A) and P15INK4b(encoded by CDKN2B): p53 induces transcription of cyclin-dependentkinase inhibitor (p21) and along with both p16INK4a and p15INK4b blockgenes for cell cycle progression (CDK4 and CDK6). This eventually leadsto hypophosphorylation of Retinoblastoma protein (Rb) and cell cyclearrest at the G1 phase.

Selectively killing senescent cells has been shown to significantlyimprove the health span of mice in the context of normal aging andameliorates the consequences of age-related disease or cancer therapy(Ovadya, J Clin Invest. 128(4):1247-1254, 2018). In nature, thesenescent cells are normally removed by the innate immune cells.Induction of senescence not only prevents the potential proliferationand transformation of damaged/altered cells, but also favors tissuerepair through the production of SASP factors that function aschemoattractants mainly for Natural Killer (NK) cells (such as IL-15 andCCL2) and macrophages (such as CFS-1 and CCL2). These innate immunecells mediate the immunosurveillance mechanism for eliminating stressedcells. Senescent cells usually up-regulate the NK-cell activatingreceptor NKG2D and DNAM-1 ligands, which belong to a family ofstress-inducible ligands: an important component of the frontline immunedefense against infectious diseases and malignancies. Upon receptoractivation, NK cells can then specifically induce the death of senescentcells through their cytolytic machinery. A role for NK cells in theimmune surveillance of senescent cells has been pointed out in liverfibrosis (Sagiv, Oncogene 32(15): 1971-1977, 2013), hepatocellularcarcinoma (Iannello, J Exp Med 210(10): 2057-2069, 2013), multiplemyeloma (Soriani, Blood 113(15): 3503-3511, 2009), and glioma cellsstressed by dysfunction of the mevalonate pathway (Ciaglia, Int J Cancer142(1): 176-190, 2018). Endometrial cells undergo acute cellularsenescence and do not differentiate into decidual cells. Thedifferentiated decidual cells secrete IL-15 and thereby recruit uterineNK cells to target and eliminate the undifferentiated senescent cellsthus helping to re-model and rejuvenate the endometrium (Brighton, Elife6: e31274, 2017). With a similar mechanism, during liver fibrosis,p53-expressing senescent liver satellite cells skewed the polarizationof resident Kupfer macrophages and freshly infiltrated macrophagestoward the pro-inflammatory M1 phenotype, which display senolyticactivity. F4/80+ macrophages have been shown to play a key role in theclearance of mouse uterine senescent cells to maintain postpartumuterine function.

Senescent cells recruit NK cells by mainly upregulating ligands to NKG2D(expressed on NK cells), chemokines, and other SASP factors. In vivomodels of liver fibrosis have shown effective clearance of senescentcells by activated NK cells (Krizhanovsky, Cell 134(4): 657-667, 2008).Studies have described various models to study senescence includingliver fibrosis (Krizhanovsky, Cell 134(4): 657-667, 2008),osteoarthritis (Xu, J Gerontol A Biol Sci Med Sci 72(6): 780-785, 2017),and Parkinson's disease (Chinta, Cell Rep 22(4): 930-940, 2018). Animalmodels for studying senescent cells are described in: Krizhanovsky, Cell134(4): 657-667, 2008; Baker, Nature 479(7372): 232-236, 2011; Farr, NatMed 23(9): 1072-1079, 2017; Bourgeois, FEBS Lett 592(12): 2083-2097,2018; Xu, Nat Med 24(8): 1246-1256, 2018).

Additional Therapeutic Agents

Some embodiments of any of the methods described herein can furtherinclude administering to a subject (e.g., any of the subjects describedherein) a therapeutically effective amount of one or more additionaltherapeutic agents. The one or more additional therapeutic agents can beadministered to the subject at substantially the same time as themulti-chain chimeric polypeptide (e.g., any of the multi-chain chimericpolypeptides described herein) or immune cell (e.g., administered as asingle formulation or two or more formulations to the subject). In someembodiments, one or more additional therapeutic agents can beadministered to the subject prior to administration of the multi-chainchimeric polypeptide (e.g., any of the multi-chain chimeric polypeptidesdescribed herein) or immune cell. In some embodiments, one or moreadditional therapeutic agents can be administered to the subject afteradministration of the multi-chain chimeric polypeptide (e.g., any of themulti-chain chimeric polypeptides described herein) or immune cell tothe subject.

Non-limiting examples of additional therapeutic agents include:anti-cancer drugs, activating receptor agonists, immune checkpointinhibitors, agents for blocking HLA-specific inhibitory receptors,Glucogen Synthase Kinase (GSK) 3 inhibitors, and antibodies.

Non-limiting examples of anticancer drugs include antimetabolic drugs(e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine,cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide,methotrexate, 6-thioguanine, cladribine, nelarabine, pentostatin, orpemetrexed), plant alkaloids (e.g., vinblastine, vincristine, vindesine,camptothecin, 9-methoxycamptothecin, coronaridine, taxol, naucleaorals,diprenylated indole alkaloid, montamine, schischkiniin, protoberberine,berberine, sanguinarine, chelerythrine, chelidonine, liriodenine,clivorine, β-carboline, antofine, tylophorine, cryptolepine,neocryptolepine, corynoline, sampangine, carbazole, crinamine,montanine, ellipticine, paclitaxel, docetaxel, etoposide, tenisopide,irinotecan, topotecan, or acridone alkaloids), proteasome inhibitors(e.g., lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib(salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770),epoxomicin, MG132, beta-hydroxy beta-methylbutyrate, bortezomib,carfilzomib, or ixazomib), antitumor antibiotics (e.g., doxorubicin,daunorubicin, epirubicin, mitoxantrone, idarubicin, actinomycin,plicamycin, mitomycin, or bleomycin), histone deacetylase inhibitors(e.g., vorinostat, panobinostat, belinostat, givinostat, abexinostat,depsipeptide, entinostat, phenyl butyrate, valproic acid, trichostatinA, dacinostat, mocetinostat, pracinostat, nicotinamide, cambinol,tenovin 1, tenovin 6, sirtinol, ricolinostat, tefinostat, kevetrin,quisinostat, resminostat, tacedinaline, chidamide, or selisistat),tyrosine kinase inhibitors (e.g., axitinib, dasatinib, encorafinib,erlotinib, imatinib, nilotinib, pazopanib, and sunitinib), andchemotherapeutic agents (e.g., all-trans retinoic acid, azacitidine,azathioprine, doxifluridine, epothilone, hydroxyurea, imatinib,teniposide, tioguanine, valrubicin, vemurafenib, and lenalidomide).Additional examples of chemotherapeutic agents include alkylatingagents, e.g., mechlorethamine, cyclophosphamide, chlorambucil,melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan,altretamine, procarbazine, dacarbazine, temozolomide, carmustine,lumustine, streptozocin, carboplatin, cisplatin, and oxaliplatin.

Non-limiting examples of activating receptor agonists include anyagonists for activating receptors which activate and enhance thecytotoxicity of NK cells, including anti-CD16 antibodies (e.g.,anti-CD16/CD30 bispecific monoclonal antibody (BiMAb)) and Fc-basedfusion proteins. Non-limiting examples of checkpoint inhibitors includeanti-PD-1 antibodies (e.g., MEDI0680), anti-PD-L1 antibodies (e.g.,BCD-135, BGB-A333, CBT-502, CK-301, CS1001, FAZ053, KN035, MDX-1105,MSB2311, SHR-1316, anti-PD-L1/CTLA-4 bispecific antibody KN046,anti-PD-L1/TGFβRII fusion protein M7824, anti-PD-L1/TIM-3 bispecificantibody LY3415244, atezolizumab, or avelumab), anti-TIM3 antibodies(e.g., TSR-022, Sym023, or MBG453) and anti-CTLA-4 antibodies (e.g.,AGEN1884, MK-1308, or an anti-CTLA-4/OX40 bispecific antibodyATOR-1015). Non-limiting examples of agents for blocking HLA-specificinhibitory receptors include monalizumab (e.g., an anti-HLA-E NKG2Ainhibitory receptor monoclonal antibody). Non-limiting examples of GSK3inhibitor include tideglusib or CHIR99021. Non-limiting examples ofantibodies that can be used as additional therapeutic agents includeanti-CD26 antibodies (e.g., YS110), anti-CD36 antibodies, and any otherantibody or antibody construct that can bind to and activate an Fcreceptor (e.g., CD16) on a NK cell. In some embodiments, an additionaltherapeutic agent can be insulin or metformin.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1. Construction of Exemplary Multi-Chain Chimeric Polypeptidesand Evaluation of Properties Thereof

Two multi-chain chimeric polypeptides were generated and theirproperties were evaluated. Each of the two multi-chain chimericpolypeptides includes a first chimeric polypeptide that includes asoluble tissue factor domain covalently linked a first target-bindingdomain and a first domain of an affinity pair of domains. The secondchimeric polypeptide in each of the two multi-chain chimericpolypeptides includes a second domain of the affinity pair of domains,and a second target-binding domain.

Description of Logic Underlying Construction of Multi-Chain ChimericPolypeptides

Tissue Factor (TF) is a stable, transmembrane protein containing 236amino acid residues. The truncated, recombinant 219-amino-acidextracellular domain of tissue factor is soluble and is known to beexpressed at high levels in bacteria or mammalian cells. Without wishingto be bound to a particular theory, the applicants speculated that the219-aa tissue factor could be used as a connector linker for creation ofunique multi-chain chimeric polypeptides.

First chimeric polypeptides including soluble tissue factor domain wereproduced at high levels by CHO cells grown in fermentation broth. Thesefirst chimeric polypeptides were purified by an anti-tissue factormonoclonal antibody (mAb) coupled on a solid matrix. Notably, tissuefactor contains binding sites for FVIIa and FX. The catalytic activityof the tissue factor-FVIIa complex for FX is approximately 1million-fold lower when tissue factor is not anchored to a phospholipidbilayer. Thus, without wishing to be bound to a particular theory,applicants speculated that using the 219-aa extracellular domain oftissue factor without the transmembrane in construction of the firstchimeric polypeptides may eliminate the pro-coagulation activity oftissue factor in the first chimeric polypeptides. In an effort tofurther reduce or eliminate the pro-coagulation activity of the 219-aatissue factor, select mutations in tissue factor can be made,specifically at seven amino acid residues that are known to contributeto binding energy of the FVIIa binding site.

Characterization of Binding Interactions for Described ChimericPolypeptides

To determine if the first and second chimeric polypeptides bind to eachother to form multi-chain chimeric polypeptides, in vitro binding assayswere performed. To determine if the first chimeric polypeptidecomprising soluble tissue factor domain are recognized and bound byanti-TF mAb, in vitro binding assays were performed. Notably, the dataindicated that the mutated tissue factor proteins are still recognizedand selectively bound by the anti-TF mAb which is known to bind to theFX binding site on tissue factor. To determine if the first chimericpolypeptides comprising soluble tissue factor domain covalently linkedto scFvs or cytokines (see FIG. 1 and FIG. 2 ) possess functional scFvsor cytokines, in vitro binding assays were performed. The data from theaforementioned assays were consistent with the purified first chimericpolypeptides having the expected biological activities (e.g. scFvsselectively bind expected target antigens or cytokines selectively bindexpected receptors or binding proteins).

In addition, experiments performed using the two multi-chain chimericpolypeptides including a first and second chimeric polypeptide bound toeach other demonstrate the expected target binding activity (e.g., themulti-chain chimeric polypeptide binds specifically to the targetspecifically recognized by the first target-binding domain and thetarget specifically recognized by the second target-binding domain).

Based on the aforementioned results, applicants concluded that thesoluble tissue factor connecter linker provided or enabled appropriatedisplay of the polypeptides encoding either scFvs, interleukins,cytokines, interleukin receptors, or cytokine receptors inthree-dimensional space relative to soluble tissue factor domain andrelative to one another such that each retained expected biologicalproperties and activities.

When both the first and second chimeric polypeptides were co-expressed,the heterodimeric complexes were secreted into the fermentation brothsat high levels. The complexes were captured and readily purified byanti-TF mAb conjugated to a solid matrix using affinity chromatography.The first and second target-binding domains of these multi-chainchimeric polypeptides retained their expected biological activities asassayed by in vitro binding assays. Thus, the assembly of themulti-chain chimeric polypeptides provides the appropriate spatialdisplay and folding of the domains for biological activities.Importantly, the spatial arrangement of the multi-chain chimericpolypeptides does not interfere with the FX binding site on tissuefactor which enables the use of anti-TF mAb for affinity purification.

Characterization of Stability for Described Chimeric Polypeptides

Both purified multi-chain chimeric polypeptides are stable. Thesemulti-chain chimeric polypeptides are structurally intact and fullybiologically active when they are incubated in human serum at 37° C. for72 hours.

Characterization of Propensity of Described Chimeric Polypeptides toAggregate

Both purified multi-chain chimeric polypeptides developed do not formaggregates when stored at 4° C. in PBS.

Characterization of Viscosity of Described Chimeric Polypeptides

There is no viscosity issue when the multi-chain chimeric polypeptidesare formulated at a concentration as high as 50 mg/mL in PBS.

Additional Applications of the Multi-Chain Chimeric Polypeptide Platform

The data from these studies show that the platform technologiesdescribed herein can be utilized to create molecules that could be fusedto target-binding domains derived from antibodies, in any of the formatsas described herein including, without limitation, adhesion molecules,receptors, cytokines, ligands, and chemokines. With the appropriatetarget-binding domain, the resulting multi-chain chimeric polypeptidescould promote conjugation of various immune effector cells and mediatedestruction of target cells, including cancer cells, virally-infectedcells, or senescent cells. Other domains in the multi-chain chimericpolypeptides stimulate, activate, and attract the immune system forenhancing cytotoxicity of effector cells for the targeted cells.

Example 2: Creation of an IL-12/IL-15RαSu DNA Construct

In a non-limiting example, an IL-12/IL-15RαSu DNA construct was created(FIG. 3 ). The human IL-12 subunit sequences, human IL-15RαSu sequence,human IL-15 sequence, human tissue factor 219 sequence, and human IL-18sequence were obtained from the UniProt website and DNA for thesesequences was synthesized by Genewiz. A DNA construct was made linkingthe IL-12 subunit beta (p40) to IL-12 subunit alpha (p35) with a GS (3)linker to generate a single chain version of IL-12 and then directlylinking the IL-12 sequence to the IL-15RαSu sequence. The finalIL-12/IL-15RαSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence of the IL12/IL-15RαSu construct (includingsignal peptide sequence) is as follows (SEQ ID NO: 77):

(Signal peptide) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCT ACTCC(Human IL-12 subunit beta (p40))ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGG GCCAGCGTGCCTTGTTCC(Linker) GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human IL-12subunit alpha (p35)) CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC (Human IL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

Example 3: Creation of an IL-18/TF/IL-15 DNA Construct

In a non-limiting example, an IL-18/TF/IL-15 construct was made (FIG. 4) linking the IL-18 sequence to the N-terminus coding region of tissuefactor 219, and further linking the IL-18/TF construct with theN-terminus coding region of IL-15. The nucleic acid sequence of theIL-18/TF/IL-15 construct (including leader sequence), synthesized byGenewiz, is as follows (SEQ ID NO: 73):

(Signal peptide) ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT ACAGC(Human IL-18) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATG TTCACCGTCCAAAACGAGGAT(Human Tissue Factor 219)AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

Example 4: Secretion of IL-12/IL-15RαSu and IL-18/TF/IL-15 FusionProteins

The IL-12/IL-15RαSu and IL-18/TF/IL-15 DNA constructs were cloned into apMSGV-1 modified retrovirus expression vector (as described by Hughes,Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), andthe expression vector was transfected into CHO-K1 cells. Co-expressionof the two constructs in CHO-K1 cells allowed for formation andsecretion of a soluble IL-18/TF/IL-15:IL-12/IL-15RαSu protein complex(referred to as 18t15-12s; FIG. 5 and FIG. 6 ). The 18t15-12s proteinwas purified from CHO-K1 cell culture supernatant using anti-TF antibodyaffinity chromatography and size exclusion chromatography resulting insoluble (non-aggregated) protein complexes consisting of IL-12/IL-15RαSuand IL-18/TF/IL-15 fusion proteins.

The amino acid sequence of the IL12/IL-15RαSu fusion protein (includingsignal peptide sequence) is as follows (SEQ ID NO: 76):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-12 subunit beta (p40))IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEW ASVPCS (Linker)GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35))RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Human IL-15R α sushidomain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The amino acid sequence of the IL-18/TF/IL-15 fusion protein (includingsignal peptide sequence) is as follows (SEQ ID NO: 72):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-18)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTVQNED (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

In some cases, the leader (signal sequence) peptide is cleaved from theintact polypeptide to generate the mature form that may be soluble orsecreted.

Example 5: Purification of 18t15-12s by Immunoaffinity Chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare™AKTA Avant protein purification system. The flow rate was 4 mL/min forall steps except the elution step, which was 2 mL/min.

Cell culture harvest of 18t15-12s was adjusted to pH 7.4 with 1M Trisbase and loaded onto the anti-TF antibody affinity column equilibratedwith 5 column volumes of PBS. After loading the sample, the column waswashed with 5 column volumes PBS, followed by elution with 6 columnvolumes 0.1M acetic acid, pH 2.9. Absorbance at 280 nm was collected andthen the sample was neutralized to pH 7.5-8.0 by adding 1M Tris base.The neutralized sample was then buffer exchanged into PBS using Amicon®centrifugal filters with a 30 KDa molecular weight cutoff. FIG. 7 showsthat the 18t15-12s complex binds the anti-TF antibody affinity column,wherein TF is an 18t15-12s binding partner. The buffer-exchanged proteinsample is stored at 2-8° C. for further biochemical analysis andbiological activity testing.

After each elution, the anti-TF antibody affinity column was thenstripped using 6 column volumes 0.1M glycine, pH 2.5. The column wasthen neutralized using 10 column volumes PBS, 0.05% sodium azide andstored at 2-8° C.

Example 6: Size Exclusion Chromatography of 18t15-12s

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration columnwas connected to a GE Healthcare AKTA™ Avant protein purificationsystem. The column was equilibrated with 2 column volumes of PBS. Theflow rate was 0.8 mL/min. A capillary loop was used to inject 2004, of 1mg/mL of 18t15-12s complex onto the column. The injection was chasedwith 1.25 column volumes of PBS. The SEC chromatograph is shown in FIG.8 . There is a main 18t15-12s protein peak with a minor high molecularweight peak, likely due to differing degrees of glycosylation of18t15-12s dimers or aggregates.

Example 7: SDS-PAGE of 18t15-12s

To determine the purity and protein molecular weight, the purified18t15-12s protein sample was analyzed using 4-12% NuPage Bis-Trisprotein gel SDS-PAGE. The gel was stained with InstantBlue™ for about 30min, followed by destaining overnight in purified water. FIG. 9 shows anexample SDS gel of anti-TF antibody affinity purified 18t15-12s, withbands at the expected molecular weights (66 kDa and 56 kDa).

Example 8: Glycosylation of 18t15-12s in CHO-K1 Cells

Glycosylation of 18t15-12s in CHO-K1 cells was confirmed using theProtein Deglycosylation Mix II kit (New England Biolabs), according tothe manufacturer's instructions. FIG. 10 shows an example SDS PAGE ofdeglycosylated and non-deglycosylated 18t15-12s. Deglycosylation reducesthe molecular weight of 18t15-12s as seen in FIG. 10 , lane 4.

Example 9: Recombinant Protein Quantitation of 18t15-12s Complexes

The 18t15-12s complex was detected and quantified using standardsandwich ELISA methods (FIGS. 11-14 ). Anti-human tissue factor antibodyserved as the capture antibody and biotinylated anti-human IL-12, IL-15,or IL-18 antibody (BAF 219, BAM 247, D045-6, all R&D Systems) served asthe detection antibody. Tissue factor in purified 18t15-12s proteincomplexes was also detected using an anti-human tissue factor captureantibody (143), and anti-human tissue factor antibody detectionantibody. The I43/anti-TF antibody ELISA was compared to purified tissuefactor at similar concentrations.

Example 10: Immunostimulatory Capacity of the 18t15-12s Complex

To assess the IL-15 immunostimulatory activity of the 18t15-12s complex,increasing concentrations of 18t15-12s was added to 32Dβ cells (104cell/well) in 200 IMDM:10% FBS media. The 32Dβ cells were incubated for3 days at 37° C. On the fourth day, WST-1 proliferation reagent (10μL/well) was added and after 4 hours, absorbance was measured at 450 nmto determine cell proliferation based on cleavage of WST-1 to a solubleformazan dye. Bioactivity of human recombinant IL-15 was assessed as apositive control. As shown in FIG. 15 , 18t15-12s demonstratedIL-15-dependent cell proliferation of 32Dβ cells. The 18t15-12s complexdemonstrated reduced activity compared to human recombinant IL-15,possibly due to the linkage of IL-18 and tissue factor to the IL-15domain.

In order to assess the individual activities of IL-12 and IL-18 in the18t15-12s complex, 18t15-12s was added to HEK-Blue IL-12 and HEK-BlueIL-18 reporter cells (5×10⁴ cell/well; hkb-il12 and hkb-hmil18,InvivoGen) in 200 μL IMDM:10% heat-inactivated FBS media. Cells wereincubated for overnight at 37° C. 20 μl of induced HEK-Blue IL-12 andHEK-Blue IL-18 reporter cell supernatant was added to 180 μl ofQUANTI-Blue (InvivoGen), and incubated for 1-3 hours at 37° C. IL-12 orIL-18 activity was assessed by measuring absorbance at 620 nm. Humanrecombinant IL-12 or IL-18 was assessed as a positive or negativecontrol. As shown in FIG. 16 and FIG. 17 , each of the cytokine domainsof the 18t15-12s complex retain specific biological activity. Theactivity of 18t15-12s was reduced compared to that of human recombinantIL-18 or IL-12, possibly due to linkage of IL-15 and tissue factor tothe IL-18 domain and linkage of IL-12 to the IL-15Rα sushi domain.

Example 11: Induction of Cytokine-Induced Memory-Like NK Cells by the18t15-12s Complex

Cytokine-induced memory-like NK cells can be induced ex vivo followingovernight stimulation of purified NK cells with saturating amounts ofIL-12 (10 ng/mL), IL-15 (50 ng/mL), and IL-18 (50 ng/mL). Thesememory-like properties have been measured through expression of IL-2receptor α (IL-2Rα, CD25), CD69 (and other activation markers), andincreased IFN-γ production. To evaluate the ability of 18t15-12scomplexes to promote generation of cytokine-induced memory-like NKcells, purified human NK cells (>95% CD56+) were stimulated for 14-18hours with 0.01 nM to 10000 nM of the 18t15-12s complex or a combinationof individual cytokines (recombinant IL-12 (10 ng/mL), IL-18 (50 ng/mL),and IL-15 (50 ng/mL)). Cell-surface CD25 and CD 69 expression andintracellular IFN-γ levels were assessed by antibody-staining and flowcytometry.

Fresh human leukocytes were obtained from a blood bank and CD56+ NKcells were isolated with the RosetteSep/human NK cell reagent (StemCellTechnologies). The purity of NK cells was >70% and confirmed by stainingwith antibodies specific to CD56-BV421, CD16-BV510, CD25-PE,CD69-APCFire750 (BioLegend). Cells were counted and resuspended in0.2×10⁶/mL in a 96 well flat bottom plate in 0.2 mL of complete media(RPMI 1640 (Gibco), supplemented with 2 mM L-glutamine (Thermo LifeTechnologies), penicillin (Thermo Life Technologies), streptomycin(Thermo Life Technologies), and 10% FBS (Hyclone)). Cells werestimulated with either a mixture of cytokines hIL-12 (10 ng/mL)(Biolegend), hIL-18 (50 ng/mL) (R&D Systems) and hIL-15 (50 ng/mL) (NCI)or with 0.01 nM to 10000 nM of the 18t15-12s at 37° C., 5% CO₂ for 14-18hrs. The cells were then harvested and surface stained with antibodiesspecific to CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 (BioLegend)for 30 minutes. After staining, cells were washed (1500 RPM for 5minutes at room temperature) in FACS buffer (1×PBS (Hyclone), with 0.5%BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After two washes,cells were analyzed using a BD FACSCelesta™ flow cytometer (PlottedData-Mean Fluorescence Intensity; FIG. 18A and FIG. 18B).

Fresh human leukocytes were obtained from a blood bank and CD56+ NKcells were isolated with the RosetteSep/human NK cell reagent (StemCellTechnologies). The purity of NK cells was >70% and confirmed by stainingwith CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specificantibodies (BioLegend). Cells were counted and resuspended in 0.2×10⁶/mLin a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640(Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies),penicillin (Thermo Life Technologies), streptomycin (Thermo LifeTechnologies), and 10% FBS (Hyclone)). Cells were stimulated with eithera cytokine mix of hIL-12 (10 ng/mL) (Biolegend), hIL-18 (50 ng/mL)(R&D), and hIL-15 (50 ng/mL) (NCI), or 0.01 nM to 10000 nM of the18t15-12s complex at 37° C., 5% CO2 for 14-18 hrs. The cells were thentreated with 10 μg/mL of Brefeldin A (Sigma) and 1× of Monensin(eBioscience) for 4 hrs before harvesting and staining with antibodiesspecific to CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 for 30minutes. After staining, cells were washed (1500 RPM for 5 minutes inroom temperature) in FACS buffer (1×PBS (Hyclone), with 0.5% BSA (EMDMillipore) and 0.001% sodium azide (Sigma)) and fixed for 10 minutes atroom temperature. After fixation, cells were washed (1500 RPM for 5minutes in room temperature) in 1× permeabilized buffer (eBioscience)and stained with IFN-γ-PE Ab (Biolegend) for 30 minutes at roomtemperature. Cells were washed once again with 1× permeabilized bufferand then washed with FACS buffer. Cell pellets were resuspended in 300μls of FACS buffer and analyzed using a BD FACSCelesta™ flow cytometer(Plotted % of IFN-γ Positive Cells; FIG. 19 ).

Example 12: In Vitro Cytotoxicity of NK Cells Against Human Tumor Cells

Human myelogenous leukemia cells, K562 (CELLTRACE®, violet dye,labelled), were incubated with purified human NK cells in the presenceof increasing concentrations of the 18t15-12s complex or a mixture ofcytokines as a control. After 20 hours, the cultures were harvested,stained with propidium iodide (PI), and assessed by flow cytometry. Asshown in FIG. 20 , the 18t15-12s complex induced human NK cytotoxicityagainst K562, at levels similar or greater than the cytokine mixture,wherein both the 18t15-12s complex and the cytokine mixture inducedgreater cytotoxicity than the medium control.

Example 13: Creation of IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15 DNAConstructs

In a non-limiting example, IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15DNA constructs were created (FIG. 21 and FIG. 22 ). The human IL-12subunit sequences, human IL-15RαSu sequence, human IL-15 sequence, humantissue factor 219 sequence, and human IL-18 sequence were synthesized byGenewiz. A DNA construct was made linking the IL-12 subunit beta (p40)to IL-12 subunit alpha (p35) with a GS (3) linker to generate a singlechain version of IL-12, directly linking the IL-12 sequence to theIL-15RαSu sequence, and directly linking the IL-12/IL-15RαSu constructto the N-terminus coding region of αCD16scFv.

The nucleic acid sequence of the IL-12/IL-15RαSu/αCD16scFv construct isas follows (SEQ ID NO: 123):

(Signal peptide) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTA CTCC(Human IL-12 subunit beta (p40))ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGG GCCAGCGTGCCTTGTTCC(Linker) GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human IL-12subunit alpha (p35)) CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC (Human IL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (anti-Human CD16 lightchain variable domain)TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT (Linker)GGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCC (anti-Human CD16 heavychain variable domain)GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAG G

Constructs were also made linking the IL-18 sequence to the N-terminuscoding region of tissue factor 219, and linking the IL-18/TF constructwith the N-terminus coding region of IL-15 (FIG. 22 ). The nucleic acidsequence of the IL-18/TF/IL-15 construct (including leader sequence) isas follows (SEQ ID NO: 73):

(Signal peptide) ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCT ACAGC(Human IL-18) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATG TTCACCGTCCAAAACGAGGAT(Human Tissue Factor 219)AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

Example 14: Secretion of IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15Fusion Proteins

The IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15 constructs were clonedinto a pMSGV-1 modified retrovirus expression vector (Hughes, Hum GeneTher 16:457-72, 2005, herein incorporated by reference), and theexpression vector was transfected into CHO-K1 cells. Co-expression ofthe two constructs in CHO-K1 cells resulted in secretion of a solubleIL-18/TF/IL-15:IL-12/IL-15RαSu/αCD16scFv protein complex (referred to as18t15-12s/αCD16; FIGS. 23 and 24 ). Co-expression of the two constructsin CHO-K1 cells resulted in secretion of the solubleIL-18/TF/IL-15:IL-12/IL-15RαSu/αCD16scFv protein complex (referred to as18t15-12s/αCD16; FIG. 23 and FIG. 24 ), which can be purified by anti-TFAb affinity and other chromatography methods. In some cases, the signalpeptide is cleaved from the intact polypeptide to generate the matureform.

The amino acid sequence of the IL-12/IL-15RαSu/αCD16scFv fusion protein(including signal peptide sequence) is as follows (SEQ ID NO: 122):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-12 subunit beta (p40))IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEW ASVPCS (Linker)GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35))RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Human IL-15R α sushidomain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (anti-Human CD16 light chain variable domain)SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGG GTKLTVGH (Linker)GGGGSGGGGSGGGGS (anti-Human CD16 heavy chain variable domain)EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGR SLLFDYWGQGTLVTVSR

The amino acid sequence of the IL-18/TF/IL-15 fusion protein (includingleader sequence) is as follows (SEQ ID NO: 72):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-18)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTVQNED (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Example 15: Creation of IL-18/IL-15RαSu and IL-12/TF/IL-15 DNAConstructs

In a non-limiting example, IL-18/IL-15RαSu and IL-12/TF/IL-15 DNAconstructs were created. The human IL-18 subunit sequences, humanIL-15RαSu sequence, human IL-12 sequence, human tissue factor 219sequence, and human IL-15 sequence were synthesized by Genewiz. A DNAconstruct was made linking IL-18 directly to IL-15RαSu. An additionalconstruct was also made linking IL-12 sequence to the N-terminus codingregion of human tissue factor 219 form, and further linking the IL-12/TFconstruct to the N-terminus coding region of IL-15. As described above,a single-chain version of IL-12 (p40-linker-p35) was used.

The nucleic acid sequence of the IL-18/IL-15RαSu construct (includingsignal peptide sequence) is as follows (SEQ ID NO: 217):

(Signal peptide) ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTA CAGC(Human IL-18) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATG TTCACCGTCCAAAACGAGGAT(Human IL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The nucleic acid sequence of the IL-12/TF/IL-15 construct (includingleader sequence) is as follows (SEQ ID NO: 218):

(Signal peptide) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTA CTCC(Human IL-12 subunit beta (p40))ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGG GCCAGCGTGCCTTGTTCC(Linker) GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human IL-12subunit alpha (p35)) CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC (Human Tissue Factor 219)AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

Example 16: Secretion of IL-18/IL-15RαSu and IL-12/TF/IL-15 FusionProteins

The IL-18/IL-15RαSu and IL-12/TF/IL-15 constructs were cloned into apMSGV-1 modified retrovirus expression vector (Hughes, Hum Gene Ther16:457-72, 2005 herein incorporated by reference), and the expressionvector was transfected into CHO-K1 cells. Co-expression of the twoconstructs in CHO-K1 cells resulted in secretion of a solubleIL-12/TF/IL-15:IL-18/IL-15RαSu protein complex (referred to as12t15/518), which can be purified by anti-TF Ab affinity and otherchromatography methods.

The amino acid sequence of the IL-18/IL-15RαSu fusion protein (includingsignal peptide sequence) is as follows (SEQ ID NO: 219):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-18)YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIM FTVQNED (Human IL-15Rα sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The amino acid sequence of the IL-12/TF/IL-15 fusion protein (includingleader sequence) is as follows (SEQ ID NO: 220):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-12 subunit beta (p40))IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEW ASVPCS (Linker)GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35))RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (Human Tissue Factor219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

Example 17: Recombinant Protein Quantitation of the 18t15-12s16 Complex

The 18t15-12s16 complex (comprising IL-12/IL-15RαSu/αCD16scFv;IL-18/TF/IL-15) was detected and quantified using standard sandwichELISA methods (FIG. 25 ). Anti-human tissue factor antibody/IL-2 oranti-TF Ab/IL-18 served as the capture antibody and biotinylatedanti-human IL-12 or IL-18 antibody (BAF 219, D045-6, both R&D Systems)served as the detection antibody. Tissue factor was also detected usingan anti-human tissue factor antibody (143), and anti-human tissue factorantibody detection antibody.

Example 18: Creation of TGFβRII/IL-15RαSu and IL-21/TF/IL-15 DNAConstructs

In a non-limiting example, a TGFβRII/IL-15RαSu DNA construct was created(FIG. 26 ). The human TGFβRII dimer and human IL-21 sequences wereobtained from the UniProt website and DNA for these sequences wassynthesized by Genewiz. A DNA construct was made linking the TGFβRII toanother TGFβRII with a linker to generate a single chain version ofTGFβRII and then directly linking the TGFβRII single chain dimersequence to the N-terminal coding region of IL-15RαSu.

The nucleic acid sequences of the TGFβRII/IL-15RαSu construct (includingsignal sequence) is as follows (SEQ ID NO: 93):

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human TGFβRII-1^(st) fragment)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT (Linker)GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT (Human TGFβRII-2^(nd)fragment) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC (HumanIL-15R α sushi domain)ATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGTATTAGA

Additionally, an IL-21/TF/IL-15 construct was made linking the IL-21sequence to the N-terminus coding region of tissue factor 219, andfurther linking the IL-21/TF construct to the N-terminus coding regionof IL-15 (FIG. 27 ). The nucleic acid sequence of the IL-21/TF/IL-15construct (including leader sequence) is as follows (SEQ ID NO: 89):

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human Tissue Factor219) TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

Example 19: Secretion of TGFβRII/IL-15RαSu and IL-21/TF/IL-15 FusionProteins

The TGFβRII/IL-15RαSu and IL-21/TF/IL-15 DNA constructs were cloned intoa pMSGV-1 modified retrovirus expression vector (as described in Hugheset al., Hum Gene Ther 16:457-72, 2005, herein incorporated byreference), and the expression vector was transfected into CHO-K1 cells.Co-expression of the two constructs in CHO-K1 cells resulted insecretion of the soluble IL-21/TF/IL-15:TGFβRII/IL-15RαSu proteincomplex (referred to as 21t15-TGFRs; FIG. 28 and FIG. 29 ). The21t15-TGFRs complex was purified from CHO-K1 cell culture supernatantusing anti-TF antibody affinity chromatography and other chromatographymethods.

The amino acid sequence of the TGFβRII/IL-15RαSu construct (includingsignal peptide sequence) is as follows (SEQ ID NO: 92):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβRII-1^(st) fragment)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Linker) GGGGSGGGGSGGGGS (HumanTGFβRII-2^(nd) fragment)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR

The amino acid sequence of the mature IL-21/TF/IL-15 fusion protein(including signal peptide sequence) is as follows (SEQ ID NO: 88):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptideto generate a mature form that may be soluble or secreted.

Example 20: Purification of 21t15-TGFRs by Immunoaffinity Chromatography

An anti-TF antibody affinity column was connected to a GE HealthcareAKTA™ Avant protein purification system. The flow rate was 4 mL/min forall steps except the elution step, which was 2 mL/min.

Cell culture harvest of 21t15-TGFRs was adjusted to pH 7.4 with 1M Trisbase and loaded onto the anti-TF antibody affinity column equilibratedwith 5 column volumes of PBS. After loading the sample, the column waswashed with 5 column volumes PBS, followed by elution with 6 columnvolumes 0.1M acetic acid, pH 2.9. Absorbance at 280 nm was collected andthen the sample was then neutralized to pH 7.5-8.0 by adding 1M Trisbase. The neutralized sample was then buffer exchanged into PBS usingAmicon® centrifugal filters with a 30 KDa molecular weight cutoff. FIG.30 shows that the 21t15-TGFRs complex binds anti-TF antibody affinitycolumn, wherein TF is a 21t15-TGFRs binding partner. Thebuffer-exchanged protein sample is stored at 2-8° C. for furtherbiochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was thenstripped using 6 column volumes 0.1M glycine, pH 2.5. The column wasthen neutralized using 10 column volumes PBS, 0.05% sodium azide, andstored at 2-8° C.

Example 21: Size Exclusion Chromatography of 21t15-TGFRs

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration columnwas connected to a GE Healthcare AKTA™ Avant protein purificationsystem. The column was equilibrated with 2 column volumes of PBS. Theflow rate was 0.8 mL/min. A capillary loop was used to inject 2004, of 1mg/mL of 21t15-TGFRs complex onto the column. The injection was thenchased with 1.25 column volumes of PBS. The SEC chromatograph was shownin FIG. 31 . There were two protein peaks, likely representing a monomerand dimer forms of 21t15-TGFRs.

Example 22: SDS-PAGE of 21t15-TGFRs

To determine the purity and protein molecular weight, the purified21t15-TGFRs complex protein sample was analyzed using 4-12% NuPageBis-Tris protein gel SDS-PAGE under reduced conditions. The gel wasstained with InstantBlue™ for about 30 min, followed by destainingovernight in purified water. FIG. 32 shows an example SDS gel of anti-TFantibody affinity purified 21t15-TGFRs, with bands at 39.08 kDa and 53kDa

Glycosylation of 21t15-TGFRs in CHO cells was confirmed using theProtein Deglycosylation Mix II kit (New England Biolabs) and themanufacturer's instructions. Deglycosylation reduces the molecularweight of 21t15-TGFRs, as seen in lane 4 of FIG. 32 .

Example 23: Recombinant Protein Quantitation of 21t15-TGFRs Complexes

The 21t15-TGFRs complex was detected and quantified using standardsandwich ELISA methods (FIGS. 33-37 ). Anti-human tissue factor antibodyserved as the capture antibody and biotinylated anti-human IL-21, IL-15,or TGFβRII served as the detection antibody. Tissue factor was alsodetected using an anti-human tissue factor capture antibody (143), andanti-human tissue factor antibody detection antibody. The I43/anti-TFantibody ELISA was compared to purified tissue factor at similarconcentrations.

Example 24: Immunostimulatory Capacity of the 21t15-TGFRs Complex

To assess the IL-15 immunostimulatory activity of the 21t15-TGFRscomplexes, increasing concentrations of 21t15-TGFRs was added to 32Dβcells (10⁴ cell/well) in 200 μL IMDM:10% FBS media and cells wereincubated for 3 days at 37° C. On the fourth day, WST-1 proliferationreagent (10 μL/well) then was added and after 4 hours, absorbance wasmeasured at 450 nm to determine cell proliferation based on cleavage ofWST-1 to a soluble formazan dye. Bioactivity of the human recombinantIL-15 was assessed as a positive control. As shown in FIG. 37 ,21t15-TGFRs demonstrated IL-15-dependent 32Dβ cell proliferation. The21t15-TGFRs complex was reduced compared to that of human recombinantIL-15, possibly due to the linkage of IL-21 and tissue factor to theIL-15 domain.

Additionally, HEK-Blue TGFβ reporter cells (hkb-tgfb, InvivoGen) wereused to measure the ability of 21t15-TGFRs to block TGFβ1 activity (FIG.38 ). Increasing concentrations of 21t15-TGFRs were mixed with 0.1 nM ofTGFβ1 and added to HEK-Blue TGFβ reporter cells (2.5×10⁴ cell/well) in200 μL IMDM:10% heat-inactivated FBS media. Cells were incubatedovernight at 37° C. The next day, 20 μl of induced HEK-Blue TGFβreporter cell supernatant was added to 180 μl of QUANTI-Blue (InvivoGen)and incubated for 1-3 hours at 37° C. 21t15-TGFRs activity was assessedby measuring absorbance at 620 nm. Human recombinant TGFβRII/Fc activitywas assessed as a positive control.

These results demonstrate that TGFβRII domain of the 21t15-TGFRs complexretains its ability to trap TGFβ1. The ability of 21t15-TGFRs to blockTGFβ1 activity was reduced compared to that of human recombinantTGFβRII/Fc, possibly due to the linkage of TGFβRII to the IL-15Rα sushidomain.

Example 25: Induction of Cytokine-Induced Memory-Like NK Cells by the21t15-TGFRs Complex

Cytokine-induced memory-like NK cells can be induced ex vivo followingovernight stimulation of purified NK cells with saturating amounts ofcytokines. These memory-like properties can be measured throughexpression of IL-2 receptor α (IL-2Rα, CD25), CD69 (and other activationmarkers), and increased IFN-γ production. To evaluate the ability of21t15-TGFRs complexes to promote generation of cytokine-inducedmemory-like NK cells, purified human NK cells (>95% CD56+) werestimulated for 14-18 hours with 1 nM to 100 nM of the 21t15-TGFRscomplex. Cell-surface CD25 and CD 69 expression and intracellular IFN-γlevels were assessed by antibody-staining and flow cytometry.

Fresh human leukocytes were obtained from a blood bank and CD56+NK cellswere isolated with the RosetteSep/human NK cell reagent (StemCellTechnologies). The purity of NK cells was >70% and confirmed by stainingwith CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specificantibodies (BioLegend). Cells were counted and resuspended in 0.2×10⁶/mLin a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640(Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies),penicillin (Thermo Life Technologies), streptomycin (Thermo LifeTechnologies), and 10% FBS (Hyclone)). Cells were stimulated with eithermix-cytokines of hIL-21 (50 ng/mL) (Biolegend) and hIL-15 (50 ng/mL)(NCI) or with 1 nM, 10 nM, or 100 nM 21t15-TGFRs complex overnight at37° C., 5% CO₂ for 14-18 hrs. The cells were then harvested and surfacestained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specificantibodies for 30 minutes. After staining, cells were washed (1500 RPMfor 5 minutes at room temperature) in FACS buffer (1×PBS (Hyclone) with0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)). After twowashes, cells were analyzed using a BD FACSCelesta™ flow cytometer.(Plotted Data-Mean Fluorescence Intensity; FIG. 39 and FIG. 40 ).

Fresh human leukocytes were obtained from a blood bank and CD56+NK cellswere isolated with the RosetteSep/human NK cell reagent (StemCellTechnologies). The purity of NK cells was >70% and confirmed by stainingwith CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specificantibodies (BioLegend). Cells were counted and resuspended in 0.2×106/mLin a 96 well flat bottom plate in 0.2 mL of complete media (RPMI 1640(Gibco), supplemented with 2 mM L-glutamine (Thermo Life Technologies),penicillin (Thermo Life Technologies), streptomycin (Thermo LifeTechnologies), and 10% FBS (Hyclone)). Cells were stimulated with eithermix-cytokines of hIL-21 (50 ng/mL) (Biolegend) and hIL-15 (50 ng/mL)(NCI) or with 1 nM, 10 nM, or 100 nM 21t15-TGFRs complex overnight at37° C., 5% CO2 for 14-18 hrs. The cells were then treated with 10 μg/mLof Brefeldin A (Sigma) and 1× of Monensin (eBioscience) for 4 hrs. Cellswere harvested and surface stained with CD56-BV421, CD16-BV510, CD25-PE,CD69-APCFire750 specific antibodies for 30 minutes. After staining,cells were washed (1500 RPM for 5 minutes at room temperature) in FACSbuffer (1×PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodiumazide (Sigma)) and fixed for 10 minutes at room temperature. Afterfixation, cells were washed (1500 RPM for 5 minutes at room temperature)with 1× permeabilized buffer (eBioscience) and stained for intracellularIFN-γ-PE Ab (Biolegend) for 30 minutes at room temperature. Cells werewashed once again with 1× permeabilized buffer and then washed with FACSbuffer. Cell pellets were resuspended in 300 μls of FACS Buffer andanalyzed using a BD FACSCelesta™ flow cytometer. (Plotted % of IFN-γPositive Cells; FIG. 41 ).

Example 26: In Vitro Cytotoxicity of NK Cells Against Human Tumor Cells

K562 (CELLTRACE®, violet dye, labelled), human myelogenous leukemiacells, were incubated with purified human NK cells (using StemCell humanNK cell purification kit (E:T ratio; 2:1)) in the presence of increasingconcentrations of the 21t15-TGFRs complex. After 20 hours, the cultureswere harvested, stained with propidium iodide (PI), and assessed by flowcytometry. As shown in FIG. 42 , the 21t15-TGFRs complex induced humanNK cytotoxicity against K562, as compared to control.

Example 27: Creation of an IL-21/TF Mutant/IL-15 DNA Construct andResulting Fusion Protein Complex with TGFβRII/IL-15RαSu

In a non-limiting example, an IL-21/TF mutant/IL-15 DNA construct wasmade by linking IL-21 directly to the N-terminus coding region of atissue factor 219 mutant, and further linking the IL-21/TF mutant to theN-terminus coding region of IL-15.

The nucleic acid sequence of the IL-21/TF mutant/IL-15 construct(including signal peptide sequence) is as follows (SEQ ID NO: 221,shaded nucleotides are mutant and the mutant codons are underlined):

(Signal sequence) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (Human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(Human Tissue Factor 219 mutants)TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCAC

ACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTC

AAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGT

TCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTC CGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of the IL-21/TF mutant/IL-15 construct(including signal peptide sequence) is as follows (SEQ ID NO: 222,substituted residues are shaded):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS  (Human Tissue Factor 219)

YTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKS TDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptideto generate a mature form that may be soluble or secreted.

In some embodiments, the IL-21/TF mutant/IL-15 DNA construct may becombined with an TGFβRII/IL-15RαSu DNA construct, transfected into cellsusing a retroviral vector as described above, and expressed as IL-21/TFmutant/IL-15 and TGFβRII/IL-15RαSu fusion proteins. The IL-15RαSu domainof the TGFβRII/IL-15RαSu fusion protein binds to the IL-15 domain of theIL-21/TF mutant/IL-15 fusion protein to create an IL-21/TFmutant/IL-15:TGFβRII/IL-15RαSu complex.

Example 28: Creation of IL-21/IL-15RαSu and TGFβRII/TF/IL-15 DNAConstructs and the Resulting Fusion Protein Complex

In a non-limiting example, an IL-21/IL-15RαSu DNA construct was made bylinking IL-21 directly to the IL-15RαSu subunit sequence. The nucleicacid sequence of the IL-21/IL-15RαSu construct (including signalsequence) is as follows (SEQ ID NO: 111):

(Signal sequence) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (Human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R α sushidomain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of the IL-21/IL-15RαSu construct (includingsignal peptide sequence) is as follows (SEQ ID NO: 110):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptideto generate a mature form that may be soluble or secreted.

In some embodiments, the IL-21/IL-15RαSu DNA construct may be combinedwith a TGFβRII/TF/IL-15 DNA construct, transfected into a retroviralvector as described above, and expressed as IL-21/IL-15RαSu andTGFβRII/TF/IL-15 fusion proteins. The IL-15RαSu domain of theIL-21/IL-15RαSu fusion protein binds to the IL-15 domain of theTGFβRII/TF/IL-15 fusion protein to create aTGFβRII/TF/IL-15:IL-21/IL-15RαSu complex.

The TGFβRII/TF/IL-15RαSu DNA construct was created by linking theTGFβRII sequence to the N-terminus coding region of human tissue factor219 form, and then linking the TGFβRII/TF construct to the N-terminuscoding region of IL-15. As described above, a single-chain version ofTGFβRII (TGFβRII-linker-TGFβRII) was used. The nucleic acid sequence ofthe TGFβRII/TF/IL-15 construct (including leader sequence) is as follows(SEQ ID NO: 136):

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human TGFβRII-1^(st) fragment)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCA ACCCTGAT (Linker)GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT (Human TGFβRII-2^(nd)fragment) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCA ACCCCGAC (HumanTissue Factor 219) TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of the TGFβRII/TF/IL-15 fusion protein(including signal peptide) is as follows (SEQ ID NO: 135):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβRII-1^(st) fragment)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Linker) GGGGSGGGGSGGGGS (HumanTGFβRII-2^(nd) fragment)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Example 29: Creation of an IL-7/IL-15RαSu DNA Construct

In a non-limiting example, an IL-7/IL-15RαSu DNA construct was created(see FIG. 43 ). The human IL-7 sequence, human IL-15RαSu sequence, humanIL-15 sequence, and human tissue factor 219 sequence were obtained fromthe UniProt website and DNA for these sequences was synthesized byGenewiz. A DNA construct was made linking the IL-7 sequence to theIL-15RαSu sequence. The final IL-7/IL-15RαSu DNA construct sequence wassynthesized by Genewiz.

The nucleic acid sequence encoding the second chimeric polypeptide ofIL-7/IL-15RαSu construct (including signal peptide sequence) is asfollows (SEQ ID NO: 103):

(Signal peptide) ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGC C(Human IL-7) GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAA GAACAC (Human IL-15Rα sushi domain) ATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGA

The second chimeric polypeptide of IL-7/IL-15RαSu construct (includingsignal peptide sequence) is as follows (SEQ ID NO: 102):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Example 30: Creation of an IL-21/TF/IL-15 DNA Construct

In a non-limiting example, an IL-21/TF/IL-15 construct was made (FIG. 44) by linking the IL-21 sequence to the N-terminus coding region oftissue factor 219, and further linking the IL-21/TF construct with theN-terminus coding region of IL-15.

The nucleic acid sequence encoding the first chimeric polypeptide ofIL-21/TF/IL-15 construct (including leader sequence), synthesized byGenewiz, is as follows (SEQ ID NO: 89):

(Signal peptide) ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGG CC(Human IL-21 fragment)CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC (Human Tissue Factor219) TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATT CAGAGAA (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide of IL-21/TF/IL-15 construct includingleader sequence is SEQ ID NO: 88:

(Signal peptide) (SEQ ID NO: 223) MGVKVLFALICIAVAEA (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Example 31: Secretion of IL-7/IL-15RαSu and IL-21/TF/IL-15 FusionProteins

The IL-7/IL-15RαSu and IL-21/TF/IL-15 DNA constructs were cloned into apMSGV-1 modified retrovirus expression vector (as described by Hughes,Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), andthe expression vector was transfected into CHO-K1 cells. Co-expressionof the two constructs in CHO-K1 cells allowed for formation andsecretion of a soluble IL-21/TF/IL-15:IL-7/IL-15RαSu protein complex(referred to as 21t15-7s; FIG. 45 and FIG. 46 ). The 21t15-7s proteinwas purified from CHO-K1 cell culture supernatant using anti-TF antibodyaffinity chromatography and size exclusion chromatography resulting insoluble (non-aggregated) protein complexes consisting of IL-7/IL-15RαSuand IL-21/TF/IL-15 fusion proteins.

In some cases, the leader (signal sequence) peptide is cleaved from theintact polypeptide to generate the mature form that may be soluble orsecreted.

Example 32: Purification of 21t15-7s by Immunoaffinity Chromatography

An anti-TF antibody affinity column was connected to a GE Healthcare™AKTA Avant protein purification system. The flow rate was 4 mL/min forall steps except the elution step, which was 2 mL/min.

Cell culture harvest of 21t15-7s was adjusted to pH 7.4 with 1M Trisbase and loaded onto the anti-TF antibody affinity column equilibratedwith 5 column volumes of PBS. After loading the sample, the column waswashed with 5 column volumes PBS, followed by elution with 6 columnvolumes 0.1M acetic acid, pH 2.9. Absorbance at 280 nm was collected andthen the sample was neutralized to pH 7.5-8.0 by adding 1M Tris base.The neutralized sample was then buffer exchanged into PBS using Amicon®centrifugal filters with a 30 KDa molecular weight cutoff. Thebuffer-exchanged protein sample was stored at 2-8° C. for furtherbiochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was thenstripped using 6 column volumes 0.1M glycine, pH 2.5. The column wasthen neutralized using 10 column volumes PBS, 0.05% sodium azide andstored at 2-8° C.

Example 33: Size Exclusion Chromatography

A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration columnwas connected to a GE Healthcare AKTA™ Avant protein purificationsystem. The column was equilibrated with 2 column volumes of PBS. Theflow rate was 0.7 mL/min. A capillary loop was used to inject 2004, of 1mg/mL of 7t15-21s complex onto the column. The injection was chased with1.25 column volumes of PBS.

Example 34: SDS-PAGE of 21t15-7s and 21t15-TGFRs

To determine the purity and protein molecular weight, the purified21t15-7s or 21t15-TGFRs protein sample were analyzed using 4-12% NuPageBis-Tris protein gel SDS-PAGE. The gel will be stained with InstantBlue™for about 30 min, followed by destaining overnight in purified water.

Example 35: Glycosylation of 21t15-7s and 21t15-TGFRs in CHO-K1 Cells

Glycosylation of 21t15-7s in CHO-K1 cells or 21t15-TGFRs in CHO-K1 cellswere confirmed using the Protein Deglycosylation Mix II kit (New EnglandBiolabs), according to the manufacturer's instructions.

Example 36: Recombinant Protein Quantitation of 21t15-7s and 21t15-TGFRsComplexes

The 21t15-7s complex or the 21t15-TGFRs complex were detected andquantified using standard sandwich ELISA methods. Anti-human tissuefactor antibody (IgG1) served as the capture antibody and biotinylatedanti-human IL-21, IL-15, or IL-7 antibody (21t15-7s) or biotinylatedanti-human IL-21, IL-15, or TGF-βRII antibody (21t15-TGFRs) served asthe detection antibody. Tissue factor in purified 21t15-7s or21t15-TGFRs protein complexes was detected using an anti-human tissuefactor capture antibody, and anti-human tissue factor antibody (IgG1)detection antibody. The anti-TF antibody ELISA will be compared topurified tissue factor at similar concentrations.

Example 37: Expansion Capacity of Primary Natural Killer (NK) Cells by21t15-7s Complex+Anti-TF IgG1 Antibody or 21t15-TGFRs Complex+Anti-TFIgG1 Antibody

To assess the 21t15-7s complex's ability to expand primary naturalkiller (NK) cells, 21t15-7s complex and 21t15-7s complex+anti-TF IgG1antibody was added to NK cells obtained from samples of fresh humanleukocytes. Cells were stimulated with 50 nM of 21t15-7s complex with orwithout 25 nM of anti-TF IgG1 or anti-TF IgG4 antibody at 37° C. and 5%CO₂. Cells were maintained at concentration at 0.5×10⁶/mL not exceeding2.0×10⁶/mL by counting every 48-72 hours and media was replenished withfresh stimulator. Cells stimulated with 21t15-7s complex or anti-TF IgG1antibody or anti-TF IgG4 antibody, or anti-TF IgG4+21t15-7s complex weremaintained up to day 5. FIG. 47 shows expansion of primary NK cells uponincubation with 21t15-7s complex+anti-TF IgG1 antibody. FIG. 54 alsoshows a schematic of the results.

Example 38: Activation of Expanded NK Cells by the 21t15-7sComplex+Anti-TF IgG1 Antibody or the 21t15-TGFRs Complex+Anti-TF IgG1Antibody

Primary NK cells can be induced ex vivo following overnight stimulationof purified NK cells with 21t15-7s complex+anti-TF IgG1 antibody. Freshhuman leukocytes were obtained from a blood bank and CD56+NK cells wereisolated with the RosetteSep/human NK cell reagent (StemCellTechnologies). The purity of NK cells was >80% and confirmed by stainingwith CD56-BV421 and CD16-BV510 specific antibodies (BioLegend). Cellswere counted and resuspended in 1×10⁶/mL in a 24 well flat bottom platein 1 mL of complete media (RPMI 1640 (Gibco), supplemented with 4 mML-glutamine (Thermo Life Technologies), penicillin (Thermo LifeTechnologies), streptomycin (Thermo Life Technologies), non-essentialamino acid (Thermo Life Technologies), sodium pyruvate (Thermo LifeTechnologies), and 10% FBS (Hyclone)). Cells were stimulated with 50 nMof 21t15-7s with or without 25 nM of anti-TF IgG1 antibody at 37° C. and5% CO₂. Cells were counted every 48-72 hours and maintained at aconcentration of 0.5×10⁶/mL to 2.0×10⁶/mL until day 14. Media wasperiodically replenished with fresh stimulator. Cells were harvested andsurface stained at day 3 with CD56-BV421, CD16-BV510, CD25-PE,CD69-APCFire750 specific antibodies (Biolegend and analyzed by FlowCytometry-Celeste-BD Bioscience). FIG. 48 shows the activation markersCD25 MFI and CD69 MFI. The activation marker CD25 MFI increased with21t15-7s complex+anti-TF IgG1 antibody stimulation, but not 21t15-7scomplex stimulation. The activation marker CD69 MFI increased with both21t15-7s complex+anti-TF IgG1 antibody and with 21t15-7s complex, alone.

Example 39: Cytotoxicity of NK Cells Against Human Tumor Cells

Fresh human blood buffy coat was obtained from a blood bank. NK cellswere isolated via negative selection using the RosetteSep/human NK cellreagent (StemCell Technologies). The NK cells were cultured in completeRPMI-1640 medium with 21t15-7s 100 nM and 50 nM of anti-TF IgG1 antibodyfor up to 11 days at 37° C. and 5% C₀2. The activated NK cells weremixed with CELLTRACE®, violet dye, labeled K562 cells at E:T ratio equalto 2:1 and incubated at 37° C. for 4 hours. The mixture was harvestedand the percentage of dead K562 cells were determined by propidiumiodide staining and flow cytometry. FIG. 49 shows increased specificlysis of K562 cells when incubated with expanded NK cells.

Example 40: Creation of an IL-21/IL-15RαSu DNA Construct

In a non-limiting example, an IL-21/IL-15RαSu DNA construct was created.The human IL-21 sequence and human IL-15RαSu sequence were obtained fromthe UniProt website and DNA for these sequences was synthesized byGenewiz. A DNA construct was made linking the IL-21 sequence to theIL-15RαSu sequence. The final IL-21/IL-15RαSu DNA construct sequence wassynthesized by Genewiz. See FIG. 50 .

Example 41: Creation of an IL-7/TF/IL-15 DNA Construct

In a non-limiting example, an IL-7/TF/IL-15 construct was made bylinking the IL-7 sequence to the N-terminus coding region of tissuefactor 219, and further linking the IL-7/TF construct with theN-terminus coding region of IL-15. See FIG. 51 .

Example 42: Creation of an IL-21/IL-15Rα Sushi DNA Construct

In a non-limiting example, a second chimeric polypeptide ofIL-21/IL-15RαSu was generated. The human IL-21 and human IL-15Rα sushisequences were obtained from the UniProt website and DNA for thesesequences was synthesized by Genewiz. A DNA construct was made linkingthe IL-21 sequence to the IL-15Rα sushi sequence. The finalIL-21/IL-15RαSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence encoding the second chimeric polypeptide ofIL-21/IL-15RαSu domain (including leader sequence), synthesized byGenewiz, is as follows (SEQ ID NO: 111):

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R α sushidomain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The second chimeric polypeptide of IL-21/IL-15Rα sushi domain (includingleader sequence) is as follows (SEQ ID NO: 110):

(Signal Sequence) MKWVTFISLLFLFSSAYS (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR

Example 43: Creation of an IL-7/TF/IL-15 DNA Construct

In a non-limiting example, an exemplary first chimeric polypeptide ofIL-7/TF/IL-15 was made by linking the IL-7 sequence to the N-terminuscoding region of tissue factor 219, and further linking the IL-7/TFconstruct with the N-terminus coding region of IL-15. The nucleic acidsequence encoding the first chimeric polypeptide of IL-7/TF/IL-15(including leader sequence), synthesized by Genewiz, is as follows (SEQID NO: 107):

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL-7 fragment) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT (Human TissueFactor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide of IL-7/TF/IL-15 (including leadersequence), is as follows (SEQ ID NO: 106):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Example 44: Secretion of IL-21/IL-15RαSu and IL-7/TF/IL-15 FusionProteins

The IL-21/IL-15RαSu and IL-7/TF/IL-15 DNA constructs were cloned into apMSGV-1 modified retrovirus expression vector (as described by Hughes,Hum Gene Ther 16:457-72, 2005, hereby incorporated by reference), andthe expression vector was transfected into CHO-K1 cells. Co-expressionof the two constructs in CHO-K1 cells allowed for formation andsecretion of a soluble IL-7/TF/IL-15:IL-21/IL-15RαSu protein complex(referred to as 7t15-21s). The 7t15-21s protein was purified from CHO-K1cell culture supernatant using anti-TF antibody (IgG1) affinitychromatography and size exclusion chromatography resulting in soluble(non-aggregated) protein complexes consisting of IL-21/IL-15RαSu andIL-7/TF/IL-15 fusion proteins. See FIG. 52 and FIG. 53 .

Example 45: Analytical Size Exclusion Chromatography (SEC) Analysis ofIL-21/IL-15RαSu and IL-7/TF/IL-15 Fusion Proteins

To determine if anti-tissue factor monoclonal antibody and 7t15-21s canform an antibody-fusion-molecule complex, analytical size exclusionchromatography (SEC) was performed. A Superdex 200 Increase 10/300 GLgel filtration column (from GE Healthcare) was connected to an AKTAAvant system (from GE Healthcare). The column was equilibrated with 2column volumes of PBS. The flow rate was 0.7 mL/min. Samples of theanti-TF mAb (1 mg/mL), 7t15-21s (1 mg/mL), and a mixture of combined ata 1:1 ratio, so the final concentration of each protein is 0.5 mg/mL)were in PBS. Each sample was injected into the Superdex 200 column usinga capillary loop, and analyzed by SEC. The SEC chromatograph of eachsample was shown in FIG. 55 . The SEC results indicated that there aretwo protein peaks for 7t15-21s, likely representing a dimer (with anapparent molecular weight of 199.2 kDa) and a higher oligomer of7t15-21s, and there is one peak (with an apparent molecular weight of206.8 kDa) for the anti-TF mAb. However, as expected, a new protein peakwith a higher molecular weight (with an apparent molecular weight of576.9 kDa) was formed in the mixture sample containing the anti-TF mAband 7t15-21s, indicating that the anti-TF mAb and 7t15-21s form anantibody-antigen complex through the binding of anti-TF mAb to TF in thefusion protein complex.

Example 46: Expansion Capacity of Primary Natural Killer (NK) Cells by7t15-21s Complex+Anti-TF IgG1 Antibody

To assess the 7t15-21s complex's ability to expand primary naturalkiller (NK) cells, 7t15-21s complex and 7t15-21s complex+anti-TF IgG1antibody are added to NK cells obtained from samples of fresh humanleukocytes. Cells are stimulated with 50 nM of 7t15-21s complex with orwithout 25 nM of anti-TF IgG1 or anti-TF IgG4 antibody at 37° C. and 5%CO₂. Cells are maintained at concentration at 0.5×10⁶/mL not exceeding2.0×10⁶/mL by counting every 48-72 hours and media is replenished withfresh stimulator. Cells stimulated with 7t15-21s complex or anti-TF IgG1antibody or anti-TF IgG4 antibody or anti-TF IgG4+7t15-21s complex aremaintained up to day 5. Expansion of primary NK cells upon incubationwith 21t15-7s complex+anti-TF IgG1 antibody is observed.

Example 47: Activation of Expanded NK Cells by the 7t15-21sComplex+Anti-TF IgG1 Antibody

Primary NK cells are induced ex vivo following overnight stimulation ofpurified NK cells with 7t15-21s complex+anti-TF IgG1 antibody. Freshhuman leukocytes are obtained from a blood bank and CD56+ NK cells areisolated with the RosetteSep/human NK cell reagent (StemCellTechnologies). The purity of NK cells is >80% and is confirmed bystaining with CD56-BV421 and CD16-BV510 specific antibodies (BioLegend).Cells are counted and resuspended in 1×10⁶/mL in a 24 well flat bottomplate in 1 mL of complete media (RPMI 1640 (Gibco), supplemented with 4mM L-glutamine (Thermo Life Technologies), penicillin (Thermo LifeTechnologies), streptomycin (Thermo Life Technologies), non-essentialamino acid (Thermo Life Technologies), sodium pyruvate (Thermo LifeTechnologies), and 10% FBS (Hyclone)). Cells are stimulated with 50 nMof 7t15-21s with or without 25 nM of anti-TF IgG1 antibody at 37° C. and5% CO₂. Cells are counted every 48-72 hours and maintained at aconcentration of 0.5×10⁶/mL to 2.0×10⁶/mL until day 14. Media isperiodically replenished with fresh stimulator. Cells are harvested andsurface stained at day 3 with CD56-BV421, CD16-BV510, CD25-PE,CD69-APCFire750 specific antibodies (Biolegend) and analyzed by FlowCytometry-Celeste-BD Bioscience). The activation marker CD25 MFI areobserved to increase with 7t15-21s complex+anti-TF IgG1 antibodystimulation, but not 7t15-21s complex stimulation. The activation markerCD69 MFI is observed to increase with both 7t15-21s complex+anti-TF IgG1antibody and with 7t15-21s complex, alone.

Example 48: Increase in Glucose Metabolism in NK Cells Using 18t15-12s

A set of experiments was performed to determine the effect of theconstruct of 18t15-12s (FIG. 6 ) on oxygen consumption rate andextracellular acidification rate (ECAR) on NK cells purified from humanblood.

In these experiments, fresh human leukocytes were obtained from theblood bank from two different human donors and NK cells were isolatedvia negative selection using the RosetteSep/human NK cell reagent(StemCell Technologies). The purity of NK cells was >80% and confirmedby staining with CD56-BV421 and CD16-BV510 specific Abs (BioLegend). Thecells were counted and resuspended in 2×10⁶/mL in 24-well, flat-bottomplates in 1 mL of complete media (RPMI 1640 (Gibco) supplemented with 4mM L-glutamine (Thermo Life Technologies), penicillin (Thermo LifeTechnologies), streptomycin (Thermo Life Technologies), non-essentialamino acid (Thermo Life Technologies), sodium pyruvate (Thermo LifeTechnologies) and 10% FBS (Hyclone)). The cells were stimulated witheither (1) media alone, (2) 100 nM 18t15-12s, or (3) mixture of singlecytokines recombinant human IL-12 (0.25 μg), recombinant human IL-15(1.25 μg), and recombinant human IL-18 (1.25 μg) overnight at 37° C. and5% CO₂. On the next day, the cells were harvested and extracellular fluxassays on expanded NK cells were performed using a XFp Analyzer(Seahorse Bioscience). The harvested cells washed and plated 2.0×10⁵cells/well in at least duplicate for extracellular flux analysis of OCR(Oxygen Consumption Rate) and ECAR (Extracellular Acidification Rate).The glycolysis stress tests were performed in Seahorse Media contain 2mM of glutamine. The following were used during the assay: 10 mMglucose; 100 nM oligomycin; and 100 mM 2-deoxy-D-glycose (2DG).

The data show that the 18t15-12s results in significantly increasedoxygen consumption rate (FIG. 56 ) and extracellular acidification rate(ECAR) as compared to the same cells activated with a combination ofrecombinant human IL-12, recombinant human IL-15, and recombinant humanIL-18 (FIG. 57 ).

Example 49: 7t15-16s21 Fusion Protein Generation and Characterization

A fusion protein complex was generated comprising ofanti-CD16scFv/IL-15RαSu/IL-21 and IL-7/TF/IL-15 fusion proteins. Thehuman IL-7 and IL-21 sequences were obtained from the UniProt websiteand DNA for these sequences was synthesized by Genewiz. Specifically, aconstruct was made linking the IL-7 sequence to the N-terminus codingregion of tissue factor 219 followed by the N-terminus coding region ofIL-15.

The nucleic acid and protein sequences of a construct comprising IL-7linked to the N-terminus of tissue factor 219 following with theN-terminus of IL-15 are shown below.

The nucleic acid sequence of the IL-7/TF/IL-15 construct (includingsignal peptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL-7) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT (Human TissueFactor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of IL-7/TF/IL-15 fusion protein (including theleader sequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Constructs were also made by linking the anti-CD16scFv sequence to theN-terminus coding region of IL-15RαSu chain followed by the N-terminuscoding region of IL-21 which was synthesized by Genewiz. The nucleicacid and protein sequences of a construct comprising the anti-CD16scFvlinked to the N-terminus of IL-15RαSu chain followed by the N-terminuscoding region of IL-21 are shown below.

The nucleic acid sequence of the anti-CD16SscFv/IL-15 RαSu/IL-21construct (including signal peptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC((Anti-human CD16scFv)TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCA CCCTGGTGACCGTGTCCAGG(Human IL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Human IL-21)CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of the anti-CD16scFv/IL-15RαSu/IL-21 construct(including signal peptide sequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Anti-human CD16scFv)SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR(Human IL-21) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

The anti-CD16scFv/IL-15RαSu/IL-21 and IL-7/TF/IL-15 constructs werecloned into a modified retrovirus expression vectors as describedpreviously (Hughes M S, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y,et al. Transfer of a TCR gene derived from a patient with a markedantitumor response conveys highly active T-cell effector functions. HumGene Ther 2005; 16:457-72), and the expression vectors were transfectedinto CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cellsallowed for formation and secretion of the solubleIL-7/TF/IL-15:anti-CD16scFv/IL-15RαSu/IL-21 protein complex (referred toas 7t15-16s21; FIG. 58 and FIG. 59 ), which can be purified by anti-TFIgG1-based affinity and other chromatography methods.

Binding of 7t15-16s21 to CHO Cells Expressing Human CD16b

CHO cells were transfected with human CD16b in a pMC plasmid andselected with 10 μg/mL of blasticidin for 10 days. The CHO cells stablyexpressing CD16b were stained with 1.2 μg/mL of 7t15-16s21, containinganti-human CD16 scFv or 18t15-12s, which does not contain anti-humanCD16 scFv, as a negative control, and then stained with biotinylatedanti-human tissue factor Ab and PE conjugated streptavidin. Onlyanti-human CD16scFv containing 7t15-16s21 stained the cells as shown inFIG. 60A. 18t15-12s did not stain the CHO cells expressing human CD16bas showed in FIG. 60B.

Detection of IL-15, IL-21, and IL-7 in 7t15-16s21 Using ELISA

A 96-well plate was coated with 100 μL (8 μg/mL) of anti-TF IgG1 in R5(coating buffer) and incubated at room temperature (RT) for 2 hrs. Theplates were washed 3 times and blocked with 100 μL of 1% BSA in PBS.Serial dilution of 7t15-16s21 (at a 1:3 ratio) were added to the wells,and incubated at RT for 60 min. Following 3 washes, 50 ng/mL ofbiotinylated-anti-IL15 antibody (BAM247, R&D Systems), 500 ng/mL ofbiotinylated-anti-IL-21 antibody (13-7218-81, R&D Systems), or 500 ng/mLof biotinylated-anti-IL-7 antibody (506602, R&D Systems) was added tothe wells and incubated at RT for 60 min. The plate was washed 3 times,and incubated with 0.25 pg/mL of HRP-SA (Jackson ImmunoResearch) at 100μL per well for 30 min at RT, followed by 4 washes and incubation with1000 of ABTS for 2 mins at RT. Absorbance was read at 405 nm. As shownin FIGS. 61A-61C, the IL-15, IL-21, and IL-7 domains in 7t15-16s21 weredetected by the individual antibodies.

The IL-15 in 7t15-16s21 Promotes IL-2Rβ and Common γ Chain Containing32Dβ Cell Proliferation

To analyze the activity of IL-15 in 7t15-16s21, the IL-15 activity of7t15-16s21 was compared to recombinant IL-15 using 32Dβ cells thatexpress IL2Rβ and common γ chain, and evaluating their effects onpromoting cell proliferation. IL-15 dependent 32Dβ cells were washed 5times with IMDM-10% FBS and seeded in the wells at 2×10⁴ cells/well.Serially-diluted 7t15-16s21 or IL-15 were added to the cells (FIG. 62 ).Cells were incubated in a CO₂ incubator at 37° C. for 3 days. Cellproliferation was detected by adding 10 μl of WST1 to each well on day 3and incubating for an additional 3 hours in a CO₂ incubator at 37° C.The absorbance at 450 nm was measured by analyzing the amount offormazan dye produced. As shown in FIG. 62 , 7t15-16s21 and IL-15promoted 32Dβ cell proliferation, with the EC₅₀ of 7t15-16s21 and IL-15being 172.2 pM and 16.63 pM, respectively.

Purification Elution Chromatograph of 7t15-16s21 from Anti-TF AntibodyAffinity Column

7t15-16s21 harvested from cell culture was loaded onto the anti-TFantibody affinity column equilibrated with 5 column volumes of PBS. Thecolumn was then washed with 5 column volumes of PBS, followed by elutionwith 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elution peakwas collected and neutralized to pH 7.5-8.0 with 1M Tris base. Theneutralized sample was buffer exchanged into PBS using Amiconcentrifugal filters with a 30 KDa molecular weight cutoff. FIG. 63 is aline graph showing the chromatographic profile of 7t15-16s21 proteincontaining cell culture supernatant following binding and elution onanti-TF antibody resin. As shown in FIG. 63 , the anti-TF antibodyaffinity column bound 7t15-16s21 which contains TF. The buffer-exchangedprotein sample was stored at 2-8° C. for further biochemical analysesand biological activity tests. After each elution, the anti-TF antibodyaffinity column was stripped using 6 column volumes of 0.1M glycine (pH2.5). The column was then neutralized using 5 column volumes of PBS, and7 column volumes of 20% ethanol for storage. The anti-TF antibodyaffinity column was connected to a GE Healthcare AKTA Avant system. Theflow rate was 4 mL/min for all steps except for the elution step, whichwas 2 mL/min.

Analytical Size Exclusion Chromatography (SEC) Analysis of 7t15-16s21

To perform size exclusion chromatography (SEC) analysis for 7t15-16s21,a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare)connected to an AKTA Avant system (GE Healthcare) was used. The columnwas equilibrated with 2 column volumes of PBS. The flow rate was 0.7mL/min. A sample containing 7t15-16s21 in PBS was injected into theSuperdex 200 column using a capillary loop, and analyzed by SEC. Asshown in FIG. 64 , the SEC results showed two protein peaks for7t15-16s21.

Example 50: TGFRt15-16s21 Fusion Protein Generation and Characterization

A fusion protein complex was generated comprising anti-humanCD16scFv/IL-15RαSu/IL21 and TGFβ Receptor II/TF/IL-15 fusion proteins(FIGS. 65 and 66 ). The human TGFβ Receptor II (Ile24-Asp159), tissuefactor 219, and IL-15 sequences were obtained from the UniProt websiteand DNA for these sequences was synthesized by Genewiz. Specifically, aconstruct was made linking two TGFβ Receptor II sequences with a G45(3)linker to generate a single chain version of TGFβ Receptor II and thendirectly linking to the N-terminus coding region of tissue factor 219followed by the N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising twoTGFβ Receptor II linked to the N-terminus of tissue factor 219 followingwith the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the two TGFβ Receptor II/TF/IL-15 construct(including signal peptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Two Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanTissue Factor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFβ Receptor II/TF/IL-15 fusion protein(including the leader sequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Constructs were also made by attaching anti-human CD16scFv directlylinking to the N-terminus coding region of IL-15RαSu chain followed bythe N-terminus coding region of IL-21 which was synthesized by Genewiz.The nucleic acid and protein sequences of a construct comprising theanti-human CD16scFv linked to the N-terminus of IL-15RαSu followed bythe N-terminus coding region of IL-21 are shown below.

The nucleic acid sequence of the anti-CD16scFv/IL-15RαSu/IL-21 construct(including signal peptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Anti-human CD16scFv) TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCA CCCTGGTGACCGTGTCCAGG(Human IL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Human IL-21)CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence of the anti-CD16scFv/IL-15RαSu/IL-21 construct(including signal peptide sequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Anti-human CD16scFv)SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR(Human IL-21) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

The anti-CD16scFv/IL-15RαSu/IL-21 and TGFR/TF/IL-15 constructs werecloned into a modified retrovirus expression vectors as describedpreviously (Hughes M S, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y,et al. Transfer of a TCR gene derived from a patient with a markedantitumor response conveys highly active T-cell effector functions. HumGene Ther 2005; 16:457-72), and the expression vectors were transfectedinto CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cellsallowed for formation and secretion of the solubleTGFR/TF/IL-15:CD16scFv/IL-15RαSu/IL-21 protein complex (referred to asTGFRt15-16s21), which can be purified by anti-TF IgG1-based affinity andother chromatography methods.

Interaction Between TGFRt15-16s21 and CHO Cells Expressing Human CD16b

CHO cells were transfected with human CD16b in a pMC plasmid andselected with 10 μg/mL of blasticidin for 10 days. Cells stablyexpressing CD16b were stained with 1.2 μg/mL of TGFRt15-16s21,containing anti-human CD16 scFv, or 7t15-21s, not containing anti-humanCD16 scFv, as a negative control, and with biotinylated anti-humantissue factor antibody and PE conjugated streptavidin. As shown in FIGS.67A and 67B, TGFRt15-16s21, which contains anti-human CD16scFv, showedpositive binding, while 7t15-21s did not show binding.

Effect of TGFRt15-16s21 on TGFβ1 Activity in HEK-Blue TGFβ Cells

To evaluate the activity of TGFβRII in TGFRt15-16s21, the effect ofTGFRt15-16s21 on the activity of TGFβ1 in HEK-Blue TGFβ cells wasanalyzed. HEK-Blue TGFβ cells (Invivogen) were washed twice withpre-warmed PBS and resuspended in the testing medium (DMEM, 10%heat-inactivated FCS, 1× glutamine, 1× anti-anti, and 2× glutamine) at5×10⁵ cells/mL. In a flat-bottom 96-well plate, 50 μl cells were addedto each well (2.5×10⁴ cells/well) and followed with 50 μL 0.1 nM TGFβ1(R&D systems). TGFRt15-16s21 or TGFR-Fc (R&D Systems) prepared at a 1:3serial dilution was then added to the plate to reach a total volume of200 μL. After 24 hrs of incubation at 37° C., 40 μL of induced HEK-BlueTGFβ cell supernatant was added to 160 μL pre-warmed QUANTI-Blue(Invivogen) in a flat-bottom 96-well plate, and incubated at 37° C. for1-3 hrs. The OD values were then determined using a plate reader(Multiscan Sky) at 620-655 nM. The IC₅₀ of each protein sample wascalculated with GraphPad Prism 7.04. The IC₅₀ of TGFRt15-16s21 andTGFR-Fc were 9127 pM and 460.6 pM respectively. These results showedthat the TGFβRII domain in TGFRt15-16s21 was able to block the activityof TGFβ-1 in HEK-Blue TGFβ cells.

The IL-15 in TGFRt15-16s21 Promotes IL-2Rβ and Common γ Chain Containing32Dβ Cell Proliferation

To analyze the activity of IL-15 in TGFRt15-16s21, the IL-15 activity ofTGFRt15-16s21 was compared to recombinant IL-15 using 32Dβ cells thatexpress IL2Rβ and common γ chain, and evaluating their effects onpromoting cell proliferation. IL-15 dependent 32Dβ cells were washed 5times with IMDM-10% FBS and seeded in the wells at 2×10⁴ cells/well.Serially-diluted TGFRt15-16s21 or IL-15 were added to the cells (FIG. 68). Cells were incubated in a CO₂ incubator at 37° C. for 3 days. Cellproliferation was detected by adding 10 μL of WST1 to each well on day 3and incubating for an additional 3 hours in a CO₂ incubator at 37° C.The absorbance at 450 nm was measured by analyzing the amount offormazan dye produced. As shown in FIG. 68 , TGFRt15-16s21 and IL-15promoted 32Eβ cell proliferation, with the EC₅₀ of TGFRt15-16s21 andIL-15 being 51298 pM and 10.63 pM, respectively.

Detection of IL-15, IL-21, and TGFβRII in TGFRt15-16s21 Using ELISA

A 96-well plate was coated with 100 μL (8 μg/mL) of anti-TF IgG1 in R5(coating buffer) and incubated at room temperature (RT) for 2 hrs. Theplates were washed 3 times and blocked with 100 μL of 1% BSA in PBS.TGFRt15-16s21 serially diluted at a 1:3 ratio was added and incubated atRT for 60 min. Following three washes, 50 ng/mL ofbiotinylated-anti-IL-15 antibody (BAM247, R&D Systems), 500 ng/mL ofbiotinylated-anti-IL-21 antibody (13-7218-81, R&D Systems), or 200 ng/mLof biotinylated-anti-TGFβRII antibody (BAF241, R&D Systems) was appliedper well, and incubated at RT for 60 min. Following three washes,incubation with 0.25 μg/mL of HRP-SA (Jackson ImmunoResearch at 100 μLper well for 30 min at RT was carried out, followed by 4 washes andincubation with 100 μL of ABTS for 2 mins at RT. Absorbance was read at405 nm. The data are shown in FIG. 69 . As shown in FIGS. 70A-70C, theIL-15, IL-21, and TGFβRII domains in TGFRt15-16s21 were detected by therespective antibodies.

Purification Elution Chromatograph of TGFRt15-16s21 Using Anti-TFAntibody Affinity Column

TGFRt15-16s21 harvested from cell culture was loaded onto the anti-TFantibody affinity column equilibrated with 5 column volumes of PBS.After sample loading, the column was washed with 5 column volumes ofPBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0with 1M Tris base. The neutralized sample was then buffer exchanged intoPBS using Amicon centrifugal filters with a 30 KDa molecular weightcutoff. As shown in FIG. 71 , the anti-TF antibody affinity column boundto TGFRt15-16s21, which contains tissue factor as a fusion partner. Thebuffer-exchanged protein sample was stored at 2-8° C. for furtherbiochemical analyses and biological activity tests. After each elution,the anti-TF antibody affinity column was stripped using 6 column volumesof 0.1M glycine (pH 2.5). The column was then neutralized using 5 columnvolumes of PBS, and 7 column volumes of 20% ethanol for storage. Theanti-TF antibody affinity column was connected to a GE Healthcare AKTAAvant system. The flow rate was 4 mL/min for all steps except for theelution step, which was 2 mL/min.

Reduced SDS-PAGE of TGFRt15-16s21

To determine the purity and molecular weight of the TGFRt15-16s21protein, protein sample purified with anti-TF antibody affinity columnwas analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPageBis-Tris gel) electrophoresis (SDS-PAGE) under reduced condition. Afterelectrophoresis, the gel was stained with InstantBlue for about 30 min,followed by destaining overnight in purified water.

To verify that the TGFRt15-16s21 protein undergoes glycosylation aftertranslation in CHO cells, a deglycosylation experiment was conductedusing the Protein Deglycosylation Mix II kit from New England Biolabsaccording to the manufacturer's instructions. FIG. 72 shows results fromthe reduced SDS-PAGE analysis of the sample in non-deglycosylated (lane1 in red outline) and deglycosylated (lane 2 in yellow outline) state.The results showed that the TGFRt15-16s21 protein is glycosylated whenexpressed in CHO cells. After deglycosylation, the purified sampleshowed expected molecular weights (69 kDa and 48 kDa) in the reduced SDSgel. Lane M was loaded with 104, of SeeBlue Plus2 Prestained Standard.

Example 51: 7t15-7s Fusion Protein Generation and Characterization

A fusion protein complex was generated comprising IL-7/TF/IL-15 andIL-7/IL-15RαSu fusion proteins (FIG. 73 and FIG. 74 ). The human IL-7,tissue factor 219, and IL-15 sequences were obtained from the UniProtwebsite and DNA for these sequences was synthesized by Genewiz.Specifically, a construct was made linking the IL-7 sequence to theN-terminus coding region of tissue factor 219 followed by the N-terminuscoding region of IL-15.

The nucleic acid and protein sequences of a construct comprising IL-7linked to the N-terminus of tissue factor 219 following with theN-terminus of IL-15 are shown below.

The nucleic acid sequence of 7t15 construct (including signal peptidesequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL7) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT (Human TissueFactor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7t15 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Constructs were also made by linking the IL-7 sequence to the N-terminuscoding region of IL-15RαSu chain which was synthesized by Genewiz. Thenucleic acid and protein sequences of a construct comprising the IL-7linked to the N-terminus of IL-15RαSu chain are shown below.

The nucleic acid sequence of 7s construct (including signal peptidesequence) is as follows:

(Signal peptide) ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGC C(Human IL7) GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAA GAACAC (Human IL-15Rα sushi domain) ATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGA

The amino acid sequence of 7s fusion protein (including the leadersequence) is as follows:

(Signal peptide) MGVKVLFALICIAVAEA (Human IL7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human IL-15Rα sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The IL-7/TF/IL-15 and IL-7/IL-15RαSu constructs were cloned into amodified retrovirus expression vectors as described previously (Hughes MS, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y, et al. Transfer of aTCR gene derived from a patient with a marked antitumor response conveyshighly active T-cell effector functions. Hum Gene Ther 2005; 16:457-72),and the expression vectors were transfected into CHO-K1 cells.Co-expression of the two constructs in CHO-K1 cells allowed forformation and secretion of the soluble IL-7/TF/IL-15:IL-7/IL-15RαSuprotein complex referred to as 7t15-7s, which can be purified by anti-TFIgG1 affinity and other chromatography methods.

Purification Elution Chromatograph of 7t15-7s Using Anti-TF AntibodyAffinity Column

7t15-7s harvested from cell culture was loaded onto the anti-TF antibodyaffinity column equilibrated with 5 column volumes of PBS. After sampleloading, the column was washed with 5 column volumes of PBS, followed byelution with 6 column volumes of 0.1M acetic acid (pH 2.9). A280 elutionpeak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base.The neutralized sample was then buffer exchanged into PBS using Amiconcentrifugal filters with a 30 KDa molecular weight cutoff. As shown inFIG. 75 , the anti-TF antibody affinity column bound to 7t15-7s whichcontains tissue factor (TF) as a fusion partner. The buffer-exchangedprotein sample was stored at 2-8° C. for further biochemical analysesand biological activity tests. After each elution, the anti-TF antibodyaffinity column was stripped using 6 column volumes of 0.1M glycine (pH2.5). The column was then neutralized using 5 column volumes of PBS, and7 column volumes of 20% ethanol for storage. The anti-TF antibodyaffinity column was connected to a GE Healthcare AKTA Avant system. Theflow rate was 4 mL/min for all steps except the elution step, which was2 mL/min.

Immunostimulation of 7t15-7s in C57BL/6 Mice

7t15-7s is a multi-chain polypeptide (a type A multi-chain polypeptidedescribed herein) that includes the first polypeptide that is a solublefusion of human IL-7, human tissue factor 219 fragment and human IL-15(7t15), and the second polypeptide that is a soluble fusion of humanIL-7 and sushi domain of human IL-15 receptor alpha chain (7s).

CHO cells were co-transfected with the IL7-TF-IL15 (7t15) and IL7-IL15Rαsushi domain (7s) vectors. The 7t15-7s complex was purified from thetransfected CHO cell culture supernatant. The IL-7, IL-15 and tissuefactor (TF) components were demonstrated in the complex by ELISA asshown in FIG. 76 . A humanized anti-TF monoclonal antibody (anti-TFIgG1) was used as the capture antibody to determine TF in 7t15-7s, andbiotinylated anti-human IL-15 antibody (R&D systems) and biotinylatedanti-human IL-7 antibody (R&D Systems) were used as the detectionantibodies to respectively detect IL-15 and IL-7 in 7t15-7s, followed byperoxidase conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTSsubstrate (Surmodics IVD, Inc.).

Example 52: TGFRt15-TGFRs Fusion Protein Generation and Characterization

A fusion protein complex was generated comprising of TGFβ ReceptorII/IL-15RαSu and TGFβ Receptor II/TF/IL-15 fusion proteins (FIG. 77 andFIG. 78 ). The human TGFβ Receptor II (Ile24-Asp159), tissue factor 219,and IL-15 sequences were obtained from the UniProt website and DNA forthese sequences was synthesized by Genewiz. Specifically, a constructwas made linking two TGFβ Receptor II sequences with a G45(3) linker togenerate a single chain version of TGFβ Receptor II and then directlylinking to the N-terminus coding region of tissue factor 219 followed bythe N-terminus coding region of IL-15.

The nucleic acid and protein sequences of a construct comprising twoTGFβ Receptor II linked to the N-terminus of tissue factor 219 followingwith the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the two TGFβ Receptor II/TF/IL-15 construct(including signal peptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Two Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanTissue Factor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFβ Receptor II/TF/IL-15 fusion protein(including the leader sequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Constructs were also made by attaching two TGFβ Receptor II directly tothe IL-15RαSu chain which was synthesized by Genewiz. The nucleic acidand protein sequences of a construct comprising the TGFβ Receptor IIlinked to the N-terminus of IL-15RαSu are shown below.

The nucleic acid sequence of the TGFβ Receptor II/IL-15RαSu construct(including signal peptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Two human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanIL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of the two TGFβ Receptor II/IL-15RαSu construct(including signal peptide sequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Two human TGFβ Receptor IIextra-cellular domains)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

The TGFβR/IL-15RαSu and TGFβR/TF/IL-15 constructs were cloned into amodified retrovirus expression vectors as described previously (Hughes MS, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y, et al. Transfer of aTCR gene derived from a patient with a marked antitumor response conveyshighly active T-cell effector functions. Hum Gene Ther 2005; 16:457-72),and the expression vectors were transfected into CHO-K1 cells.Co-expression of the two constructs in CHO-K1 cells allowed forformation and secretion of the soluble TGFβR/TF/IL-15:TGFβR/IL-15RαSuprotein complex (referred to as TGFRt15-TGFRs), which can be purified byanti-TF IgG1 affinity and other chromatography methods.

Effect of TGFRt15-TGFRs on TGFβ1 Activity in HEK-Blue TGFβ Cells

To evaluate the activity of TGFβRII in TGFRt15-TGFRs, the effect ofTGFRt15-TGFRs on the activity of TGFβ1 in HEK-Blue TGFβ cells wasanalyzed. HEK-Blue TGFβ cells (Invivogen) were washed twice withpre-warmed PBS and resuspended in the testing medium (DMEM, 10%heat-inactivated FCS, 1× glutamine, 1× anti-anti, and 2× glutamine) at5×10⁵ cells/mL. In a flat-bottom 96-well plate, 50 μL cells were addedto each well (2.5×10⁴ cells/well) and followed with 50 μL 0.1 nM TGFβ1(R&D systems). TGFRt15-TGFRs or TGFR-Fc (R&D Systems) prepared at a 1:3serial dilution was then added to the plate to reach a total volume of200 μL. After 24 hrs of incubation at 37° C., 40 μL of induced HEK-BlueTGFβ cell supernatant was added to 160 μL pre-warmed QUANTI-Blue(Invivogen) in a flat-bottom 96-well plate, and incubated at 37° C. for1-3 hrs. The OD values were then determined using a plate reader(Multiscan Sky) at 620-655 nM. The IC₅₀ of each protein sample wascalculated with GraphPad Prism 7.04. The IC₅₀ of TGFRt15-TGFRs andTGFR-Fc were 216.9 pM and 460.6 pM respectively. These results showedthat the TGFβRII domain in TGFRt15-TGFRs was able to block the activityof TGFβ1 in HEK-Blue TGFβ cells.

The IL-15 in TGFRt15-TGFRs Promotes IL-2Rβ and Common γ Chain Containing32Dβ Cell Proliferation

To evaluate the activity of IL-15 in TGFRt15-TGFRs, the IL-15 activityof TGFRt15-TGFRs was compared to recombinant IL-15 using 32Dβ cells thatexpress IL2Rβ and common γ chain, and evaluating their effects onpromoting cell proliferation. IL-15 dependent 32Dβ cells were washed 5times with IMDM-10% FBS and seeded in the wells at 2×10⁴ cells/well.Serially-diluted TGFRt15-TGFRs or IL-15 were added to the cells (FIG. 79). Cells were incubated in a CO₂ incubator at 37° C. for 3 days. Cellproliferation was detected by adding 10 μL of WST1 to each well on day 3and incubating for an additional 3 hours in a CO₂ incubator at 37° C.The absorbance at 450 nm was measured by analyzing the amount offormazan dye produced. As shown in FIG. 79 , TGFRt15-TGFRs and IL-15promoted 32Dβ cell proliferation, with the EC₅₀ of TGFRt15-TGFRs andIL-15 being 1901 pM and 10.63 pM, respectively.

Detection of IL-15 and TGFβRII Domains in TGFRt15-TGFRs withCorresponding Antibodies Using ELISA

A 96-well plate was coated with 100 μL (8 μg/mL) of anti-TF IgG1 in R5(coating buffer) and incubated at room temperature (RT) for 2 hrs. Theplates were washed 3 times and blocked with 100 μL of 1% BSA in PBS.TGFRt15-TGFRs was added at a 1:3 serial dilution, and incubated at RTfor 60 min. After 3 washes, 50 ng/mL of biotinylated-anti-IL-15 antibody(BAM247, R&D Systems), or 200 ng/mL of biotinylated-anti-TGFβRIIantibody (BAF241, R&D Systems) was added to the wells and incubated atRT for 60 min. Next the plates were washed 3 times, and 0.25 μg/mL ofHRP-SA (Jackson ImmunoResearch) at 100 μL per well was added andincubated for 30 min at RT, followed by 4 washes and incubation with 100μL of ABTS for 2 mins at RT. Absorbance at 405 nm was read. As shown inFIGS. 81A and 81B, the IL-15 and TGFβRII domains in TGFRt15-TGFRs weredetected by the individual antibodies.

Purification Elution Chromatograph of TGFRt15-TGFRs from Anti-TFAntibody Affinity Column

TGFRt15-TGFRs harvested from cell culture was loaded onto the anti-TFantibody affinity column equilibrated with 5 column volumes of PBS.After sample loading, the column was washed with 5 column volumes ofPBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0with 1M Tris base. The neutralized sample was then buffer exchanged intoPBS using Amicon centrifugal filters with a 30 KDa molecular weightcutoff. As shown in FIG. 82 , the anti-TF antibody affinity column boundto TGFRt15-TGFRs which contains TF as a fusion partner. Thebuffer-exchanged protein sample was stored at 2-8° C. for furtherbiochemical analyses and biological activity tests. After each elution,the anti-TF antibody affinity column was stripped using 6 column volumesof 0.1M glycine (pH 2.5). The column was then neutralized using 5 columnvolumes of PBS, and 7 column volumes of 20% ethanol for storage. Theanti-TF antibody affinity column was connected to a GE Healthcare AKTAAvant system. The flow rate was 4 mL/min for all steps except for theelution step, which was 2 mL/min.

Analytical Size Exclusion Chromatography (SEC) Analysis of TGFRt15-TGFRs

A Superdex 200 Increase 10/300 GL gel filtration column (from GEHealthcare) was connected to an AKTA Avant system (from GE Healthcare).The column was equilibrated with 2 column volumes of PBS. The flow ratewas 0.7 mL/min. A sample containing TGFRt15-TGFRs in PBS was injectedinto the Superdex 200 column using a capillary loop, and analyzed bySEC. The SEC chromatograph of the sample is shown in FIG. 83 . The SECresults showed four protein peaks for TGFRt15-TGFRs.

Reduced SDS-PAGE Analysis of TGFRt15-TGFRs

To determine the purity and molecular weight of the TGFRt15-TGFRsprotein, protein sample purified with anti-TF antibody affinity columnwas analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPageBis-Tris gel) electrophoresis (SDS-PAGE) method under reduced condition.After electrophoresis, the gel was stained with InstantBlue for about 30min, followed by destaining overnight in purified water.

To verify that the TGFRt15-TGFRs protein undergoes glycosylation aftertranslation in CHO cells, a deglycosylation experiment was conductedusing the Protein Deglycosylation Mix II kit from New England Biolabsand the manufacturer's instructions. FIG. 84 shows the reduced SDS-PAGEanalysis of the sample in non-deglycosylated (lane 1 in red outline) anddeglycosylated (lane 2 in yellow outline) state. The results showed thatthe TGFRt5-TGFRs protein is glycosylated when expressed in R) cells.After deglycosylation, the purified sample showed expected molecularweights (69 kDa and 39 kDa) in the reduced SDS gel. Lane M was loadedwith 10 ul of SeeBlue Plus2 Prestained Standard.

Immunostimulatory Activity of TGFRt15-TGFRs in C57BL/6 Mice

TGFRt15-TGFRs is a multi-chain polypeptide (a type A multi-chainpolypeptide described herein) that includes a first polypeptide that isa soluble fusion of two TGFβRII domains, human tissue factor 219fragment and human IL-15, and the second polypeptide that is a solublefusion of two TGFβRII domains and sushi domain of human IL-15 receptoralpha chain.

Wild type C57BL/6 mice were treated subcutaneously with either controlsolution or with TGFRt15-TGFRs at a dosage of 0.3 mg/kg, 1 mg/kg, 3mg/kg, or 10 mg/kg. Four days after treatment, spleen weight and thepercentages of various immune cell types present in the spleen wereevaluated. As shown in FIG. 85A, the spleen weight in mice treated withTGFRt15-TGFRs increased with increasing dosage of TGFRt15-TGFRs.Moreover, the spleen weight in mice treated with 1 mg/kg, 3 mg/kg, and10 mg/kg of TGFRt15-TGFRs were higher as compared to mice treated withthe control solution, respectively. In addition, the percentages of CD4⁺T cells, CD8⁺ T cells, NK cells, and CD19⁺ B cells present in the spleenof control-treated and TGFRt15-TGFRs-treated mice were evaluated. Asshown in FIG. 85B, in the spleens of mice treated with TGFRt15-TGFRs,the percentages of CD8⁺ T cells and NK cells both increased withincreasing dosage of TGFRt15-TGFRs. Specifically, the percentages ofCD8⁺ T cells were higher in mice treated with 0.3 mg/kg, 3 mg/kg, and 10mg/kg of TGFRt15-TGFRs compared to control-treated mice, and thepercentages of NK cells were higher in mice treated with 0.3 mg/kg, 1mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs compared tocontrol-treated mice. These results demonstrate that TGFRt15-TGFRs isable to stimulate immune cells in the spleen, in particular CD8⁺ T cellsand NK cells.

The pharmacokinetics of TGFRt15-TGFRs molecules were evaluated in wildtype C57BL/6 mice. The mice were treated subcutaneously withTGFRt15-TGFRs at a dosage of 3 mg/kg. The mouse blood was drained fromtail vein at various time points and the serum was prepared. TheTGFRt15-TGFRs concentrations in mouse serum was determined with ELISA(capture: anti-human tissue factor antibody; detection: biotinylatedanti-human TGFβ receptor antibody and followed by peroxidase conjugatedstreptavidin and ABTS substrate). The results showed that the half-lifeof TGFRt15-TGFRs was 12.66 hours in C57BL/6 mice.

The mouse splenocytes were prepared in order to evaluate theimmunostimulatory activity of TGFRt15-TGFRs over time in mice. As shownin FIG. 86A, the spleen weight in mice treated with TGFRt15-TGFRsincreased 48 hours posttreatment and continued to increase over time. Inaddition, the percentages of CD4⁺ T cells, CD8⁺ T cells, NK cells, andCD19⁺ B cells present in the spleen of control-treated andTGFRt15-TGFRs-treated mice were evaluated. As shown in FIG. 86B, in thespleens of mice treated with TGFRt15-TGFRs, the percentages of CD8⁺ Tcells and NK cells both increased at 48 hours after treatment and werehigher and higher overtime after the single dose treatment. Theseresults further demonstrate that TGFRt15-TGFRs is able to stimulateimmune cells in the spleen, in particular CD8⁺ T cells and NK cells.

Furthermore, the dynamic proliferation of immune cells based on Ki67expression of splenocytes and cytotoxicity potential based on granzyme Bexpression were evaluated in splenocytes isolated from mice following asingle dose (3 mg/kg) of TGFRt15-TGFRs. As shown in FIGS. 87A and 87B,in the spleens of mice treated with TGFRt15-TGFRs, the expression ofKi67 and granzyme B by NK cells increased at 24 hours after treatmentand its expression of CD8⁺ T cells and NK cells both increased at 48hours and later time points after the single dose treatment. Theseresults demonstrate that TGFRt15-TGFRs not only increases the numbers ofCD8⁺ T cells and NK cells but also enhance the cytotoxicity of thesecells. The single dose treatment of TGFRt15-TGFRs led CD8⁺ T cells andNK cells to proliferate for at least 4 days.

The cytotoxicity of the splenocytes from TGFRt15-TGFRs-treated miceagainst tumor cells was also evaluated. Mouse Moloney leukemia cells(Yac-1) were labeled with CELLTRACE®, violet dye, and were used as tumortarget cells. Splenocytes were prepared from TGFRt15-TGFRs (3mg/kg)-treated mouse spleens at various time points post treatment andwere used as effector cells. The target cells were mixed with effectorcells at an E:T ratio=10:1 and incubated at 37° C. for 20 hours. Targetcell viability was assessed by analysis of propidium iodide positive,violet-labeled Yac-1 cells using flow cytometry. Percentage of Yac-1tumor inhibition was calculated using the formula, (1−[viable Yac-1 cellnumber in experimental sample]/[viable Yac-1 cell number in the samplewithout splenocytes])×100. As shown in FIG. 88 , splenocytes fromTGFRt15-TGFRs-treated mice had stronger cytotoxicity against Yac-1 cellsthan the control mouse splenocytes.

Tumor Size Analysis in Response to Chemotherapy and/or TGFRt15-TGFRs

Pancreatic cancer cells (SW1990, ATCC® CRL-2172) were subcutaneously(s.c.) injected into C57BL/6 scid mice (The Jackson Laboratory, 001913,2×10⁶ cells/mouse, in 100 μL HBSS) to establish the pancreatic cancermouse model. Two weeks after tumor cell injection, chemotherapy wasinitiated in these mice intraperitoneally with a combination of Abraxane(Celgene, 68817-134, 5 mg/kg, i.p.) and Gemcitabine (Sigma Aldrich,G6423, 40 mg/kg, i.p.), followed by immunotherapy with TGFRt15-TGFRs (3mg/kg, s.c.) in 2 days. The procedure above was considered one treatmentcycle and was repeated for another 3 cycles (1 cycle/week). Controlgroups were set up as the SW1990-injected mice that received PBS,chemotherapy (Gemcitabine and Abraxane), or TGFRt15-TGFRs alone. Alongwith the treatment cycles, tumor size of each animal was measured andrecorded every other day, until the termination of the experiment 2months after the SW1990 cells were injected. Measurement of the tumorvolumes were analyzed by group and the results indicated that theanimals receiving a combination of chemotherapy and TGFRt15-TGFRs hadsignificantly smaller tumors comparing to the PBS group, whereas neitherchemotherapy nor TGFRt15-TGFRs therapy alone work as sufficiently as thecombination (FIG. 89 ).

In Vitro Senescent B16F10 Melanoma Model

Next, in vitro killing of senescent B16F10 melanoma cells by activatedmouse NK cells was evaluated. B16F10 senescence cells (B16F10-SNC) cellswere labelled with CELLTRACE®, violet dye, and incubated for 16 hrs withdifferent E:T ratio of in vitro 2t2-activated mouse NK cells (isolatedfrom spleen of C57BL/6 mice injected with TGFRt15-TGFRs10 mg/kg for 4days). The cells were trypsinized, washed and resuspended in completemedia containing propidium iodide (PI) solution. The cytotoxicity wasassessed by flow cytometry (FIG. 90 ).

Example 53: 7t15-21s137L (Long Version) Fusion Protein Creation andCharacterization

A fusion protein complex was generated comprising ofIL-21/IL-15RαSu/CD137L and IL-7/TF/IL-15 fusion proteins (FIG. 91 andFIG. 92 ). Specifically, a construct was made linking the IL-7 sequenceto the N-terminus coding region of tissue factor 219 followed by theN-terminus coding region of IL-15. The nucleic acid and proteinsequences of a construct comprising IL-7 linked to the N-terminus oftissue factor 219 following with the N-terminus of IL-15 are shownbelow.

The nucleic acid sequence of the 7t15 construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL7) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT (Human TissueFactor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7t15 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

The nucleic acid and protein sequences of the 21s137L are shown below.The nucleic acid sequence of the 21s137L construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R α sushidomain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGG AA

The amino acid sequence of 21s137L fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR((G4S)3 linker) GGGGSGGGGSGGGGS (Human CD137L)REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

The IL-21/IL-15RαSu/CD137L and IL-7/TF/IL-15 constructs were cloned intoa modified retrovirus expression vectors as described previously (HughesM S, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y, et al. Transfer ofa TCR gene derived from a patient with a marked antitumor responseconveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1cells. Co-expression of the two constructs in CHO-K1 cells allowed forformation and secretion of the soluble IL-7/TF/IL-15:IL-21/IL-15RαSu/CD137L protein complex (referred to as 7t15-21s137L),which can be purified by anti-TF IgG1 affinity and other chromatographymethods.

Purification Elution Chromatograph of 7t15-21s137L Using Anti-TFAntibody Affinity Column

7t15-21s137L harvested from cell culture was loaded onto the anti-TFantibody affinity column equilibrated with 5 column volumes of PBS.After sample loading, the column was washed with 5 column volumes ofPBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0with 1M Tris base. The neutralized sample was then buffer exchanged intoPBS using Amicon centrifugal filters with a 30 KDa molecular weightcutoff. As shown in FIG. 93 , the anti-TF antibody affinity column boundto 7t15-21s137L which contains TF as a fusion partner. Thebuffer-exchanged protein sample was stored at 2-8° C. for furtherbiochemical analyses and biological activity tests. After each elution,the anti-TF antibody affinity column was stripped using 6 column volumesof 0.1M glycine (pH 2.5). The column was then neutralized using 5 columnvolumes of PBS, and 7 column volumes of 20% ethanol for storage. Theanti-TF antibody affinity column was connected to a GE Healthcare AKTAAvant system. The flow rate was 4 mL/min for all steps except for theelution step, which was 2 mL/min. FIG. 94 shows the analytical SECprofile of 7t15-21s137L.

Example 54: 7t15-21s137L (Short Version) Fusion Protein Generation andCharacterization

A fusion protein complex was generated comprising ofIL-21/IL-15RαSu/CD137L and IL-7/TF/IL-15 fusion proteins. Specifically,a construct was made linking the IL-7 sequence to the N-terminus codingregion of tissue factor 219 followed by the N-terminus coding region ofIL-15. The nucleic acid and protein sequences of a construct comprisingIL-7 linked to the N-terminus of tissue factor 219 following with theN-terminus of IL-15 are shown below.

The nucleic acid sequence of 7t15 construct (including signal peptidesequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL7) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT (Human TissueFactor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7t15 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

The nucleic acid and protein sequences of the 21s137L (short version)are shown below. The nucleic acid sequence of 21s137L (short version)construct (including signal peptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R α sushidomain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137 Ligand shortversion) GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC

The amino acid sequence of the 21s137L (short version) construct(including signal peptide sequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIR((G4S)3 linker) GGGGSGGGGSGGGGS (Human CD137 Ligand short version)DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT QGATVLGLFRVTPEI

The IL-21/IL-15RαSu/CD137L (short version) and IL-7/TF/IL-15 constructswere cloned into a modified retrovirus expression vectors as describedpreviously (Hughes M S, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y,et al. Transfer of a TCR gene derived from a patient with a markedantitumor response conveys highly active T-cell effector functions. HumGene Ther 2005; 16:457-72), and the expression vectors were transfectedinto CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cellsallowed for formation and secretion of the soluble IL-7/TF/IL-15:IL-21/IL-15RαSu/CD137L protein complex (referred to as 7t15-21s137L(short version)), which can be purified by anti-TF IgG1 affinity andother chromatography methods.

Binding of 7t15-21s137L (short version) to CD137 (4.1BB)

On day 1, a 96-well plate was coated with 100 μL (2.5 μg/mL) of GAH IgGFc (G-102-C, R&D Systems) in R5 (coating buffer) or R5 only andincubated at 4° C., overnight. On day 2, the plates were washed threetimes and blocked with 300 μL of 1% BSA in PBS at 37° C. for 2 hrs. 10ng/mL of 4.1BB/Fc (838-4B, R&D Systems) was added at 100 μL/well andincubated for 2 hrs at RT. After three washes, the 7t15-21s137L or7t15-21s serially diluted at a 1/3 ratio (starting at 10 nM), andincubated at 4° C. overnight. On day 3, following 3 washes, 300 ng/mL ofbiotinylated-anti-hTF antibody (BAF2339, R&D Systems) was added at 100μL per well and incubated at RT for 2 hrs. The plate was then washedthree times and incubated with 0.25 μg/mL of HRP-SA (JacksonImmuneResearch) at 100 μL per well for 30 min, followed by 3 washes andincubation with 100 μL of ABTS for 2 mins at RT. Absorbance was read at405 nm. As shown in FIG. 95 , 7t15-21s137L (short version) showedsignificant interaction with 4.1BB/Fc (blue line) as compared to7t15-21s.

Detection of IL-15, IL-21, and IL-7 in 7t15-21s137L (Short Version) withELISA

A 96-well plate was coated with 100 μL (8 μg/mL) of anti-TF IgG1 in R5(coating buffer) and incubated at RT for 2 hrs. The plates were washed 3times and blocked with 100 μL of 1% BSA in PBS. 7t15-21s137L (shortversion), serially diluted at a 1:3 ratio was added, and incubated at RTfor 60 min. After three washes, 50 ng/mL of biotinylated-anti-IL15antibody (BAM247, R&D Systems), 500 ng/mL of biotinylated-anti-IL21antibody (13-7218-81, R&D Systems), or 500 ng/mL ofbiotinylated-anti-IL7 antibody (506602, R&D Systems) was added to thewells and incubated at RT for 60 min. After three washes and incubationwith 0.25 μg/mL of HRP-SA (Jackson ImmunoResearch) at 100 μL per wellwas carried out for 30 min at RT, followed by four washes and incubationwith 100 μL of ABTS for 2 mins at RT. Absorbance was read at 405 nm. Asshown in FIGS. 96A-96C, the IL-15, IL-21, and IL-7 domains in7t15-21s137L (short version) were detected by the respective antibodies.

The IL-15 in 7t15-1s137L (Short Version) Promotes IL2Rαβγ ContainingCTLL2 Cell Proliferation

To evaluate the IL-15 activity of 7t15-21s137L (short version),7t15-21s137L (short version) was compared with recombinant IL15 inpromoting proliferation of IL2Rαβγ expressing CTLL2 cells.IL-15-dependent CTLL2 cells were washed 5 times with IMDM-10% FBS andseeded to the wells at 2×10⁴ cells/well. Serially diluted 7t15-21s137L(short version) or IL-15 were added to the cells (FIG. 97 ). Cells wereincubated in a CO₂ incubator at 37° C. for 3 days. Cell proliferationwas detected by adding 10 μL of WST1 to each well on day 3 and incubatedfor an additional 3 hours in a CO₂ incubator at 37° C. The amount offormazan dye produced was analyzed by measuring the absorbance at 450nm. As shown in FIG. 97 , 7t15-21s137L (short version) and IL-15promoted CTLL2 cell proliferation. The EC₅₀ of 7t15-21s137L (shortversion) and IL-15 was 55.91 pM and 6.22 pM. respectively.

The IL-21 in 7t15-1s137L (Short Version) Promotes IL21R Containing B9Cell Proliferation

To evaluate the IL-21 activity of 7t15-21s137L (short version),7t15-21s137L (short version) was compared with recombinant IL-21 inpromoting proliferation of IL-21R expressing B9 cells. IL-21R containingB9 cells were washed 5 times with RPMI-10% FBS and seeded to the wellsat 1×10⁴ cells/well. Serially diluted 7t15-21s137L (short version) orIL-21 were added to the cells (FIG. 98 ). Cells were incubated in a CO₂incubator at 37° C. for 5 days. Cell proliferation was detected byadding 10 μL of WST1 to each well on day 5 and incubated for anadditional 4 hours in a CO₂ incubator at 37° C. The amount of formazandye produced was analyzed by measuring the absorbance at 450 nm. Asshown in FIG. 98 , 7t15-21s137L (short version) and IL-21 promoted B9cell proliferation. The EC₅₀ of 7t15-21s137L (short version) and IL-21was 104.1 nM and 72.55 nM, respectively.

Example 55: 7t15-TGFRs Fusion Protein Generation and Characterization

A fusion protein complex was generated comprising of TGFβ ReceptorII/IL-15RαSu and IL-7/TF/IL-15 fusion proteins (FIG. 99 and FIG. 100 ).The human TGFβ Receptor II (Ile24-Asp159), tissue factor 219, IL-15, andIL-7 sequences were obtained from the UniProt website and DNA for thesesequences was synthesized by Genewiz. Specifically, a construct was madelinking the IL-7 sequence to the N-terminus coding region of tissuefactor 219 followed by the N-terminus coding region of IL-15. Thenucleic acid and protein sequences of a construct comprising IL-7 linkedto the N-terminus of tissue factor 219 following with the N-terminus ofIL-15 are shown below.

The nucleic acid sequence of the 7t15 construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC(Human IL7) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT (Human TissueFactor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7t15 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Constructs were also made by attaching two TGFβ Receptor II directly tothe IL-15RαSu chain which was synthesized by Genewiz. The nucleic acidand protein sequences of a construct comprising the TGFβ Receptor IIlinked to the N-terminus of IL-15RαSu are shown below.

The nucleic acid sequence of the TGFRs construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC(Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanIL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence of TGFRs fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIR

Effect of 7t15-TGFRs on TGFβ1 Activity in HEK-Blue TGFβ Cells

To evaluate the activity of TGFβR in 7t15-TGFRs, the effect of7t15-TGFRs on the activity of TGFβ1 in HEK-Blue TGFβ cells was analyzed.HEK-Blue TGFβ cells (Invivogen) were washed twice with pre-warmed PBSand resuspended in the testing medium (DMEM, 10% heat-inactivated FCS,1× glutamine, 1× anti-anti, and 2× glutamine) at 5×10⁵ cells/mL. In aflat-bottom 96-well plate, 50 μL cells were added to each well (2.5×10⁴cells/well) and followed with 50 μL 0.1 nM TGFβ1 (R&D systems).7t15-TGFRs or TGFR-Fc (R&D Systems) prepared at a 1:3 serial dilutionwas then added to the plate to reach a total volume of 200 μL. After 24hrs of incubation at 37° C., 40 μL of induced HEK-Blue TGFβ cellsupernatant was added to 160 μL pre-warmed QUANTI-Blue (Invivogen) in aflat-bottom 96-well plate, and incubated at 37° C. for 1-3 hrs. The ODvalues were then determined using a plate reader (Multiscan Sky) at620-655 nM. The IC₅₀ of each protein sample was calculated with GraphPadPrism 7.04. The IC₅₀ of 7t15-TGFRs and TGFR-Fc were 1142 pM and 558.6 pMrespectively. These results showed that the TGFβR in 7t15-TGFRs was ableto block the activity of TGFβ1 in HEK-Blue TGFβ cells.

Detection of IL-15, TGFβRII, and IL-7 in 7t15-TGFRs with ELISA

A 96-well plate was coated with 100 μL (8 μg/mL) of anti-TF IgG1 in R5(coating buffer) and incubated at room temperature (RT) for 2 hrs. Theplates were washed three times and blocked with 100 μL of 1% BSA in PBS.Serial dilution of 7t15-TGFRs (1:3 ratio) was added, and incubated at RTfor 60 mins. After 3 washes, 50 ng/mL of biotinylated-anti-IL-15antibody (BAM247, R&D Systems), 200 ng/mL of biotinylated-anti-TGFbRIIantibody (BAF241, R&D Systems), or 500 ng/mL of biotinylated-anti-IL-7antibody (506602, R&D Systems) was added and incubated at RT for 60 min.Following three washes, incubation with 0.25 μg/mL of HRP-SA (JacksonImmunoResearch) at 100 μL per well was carried out for 30 min at RT,followed by 4 washes and incubation with 100 μL of ABTS for 2 mins atRT. Absorbance was read at 405 nm. The data are shown in FIG. 101 . Asshown in FIGS. 102A-102C, the IL-15, TGFR, and IL-7 in 7t15-TGFRs weredetected by the respective antibodies.

The IL-15 in 7t15-TGFRs Promotes IL-2Rβ and Common γ Chain Containing32Dβ Cell Proliferation

To evaluate the activity of IL-15 in 7t15-TGFRs, 7t15-TGFRs was comparedto recombinant IL-15 using 32Dβ cells that express IL2Rβ and common γchain, and evaluating their effects on promoting cell proliferation.IL-15 dependent 32Dβ cells were washed 5 times with IMDM-10% FBS andseeded in the wells at 2×10⁴ cells/well. Serially-diluted 7t15-TGFRs orIL-15 were added to the cells (FIG. 103 ). Cells were incubated in a CO₂incubator at 37° C. for 3 days. Cell proliferation was detected byadding 10 μL of WST1 to each well on day 3 and incubating for anadditional 3 hours in a CO₂ incubator at 37° C. The amount of formazandye produced was analyzed by measuring the absorbance at 450 nm. Asshown in FIG. 103 , 7t15-TGFRs and IL-15 promoted 32Dβ cellproliferation, with the EC₅₀ of 7t15-TGFRs and IL-15 being 126 nM and16.63 pM, respectively.

Purification Elution Chromatograph of 7t15-TGFRs Using Anti-TF AntibodyAffinity Column

7t15-TGFRs harvested from cell culture was loaded onto the anti-TFantibody affinity column equilibrated with 5 column volumes of PBS.After sample loading, the column was washed with 5 column volumes ofPBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0with 1M Tris base. The neutralized sample was then buffer exchanged intoPBS using Amicon centrifugal filters with a 30 KDa molecular weightcutoff. As shown in FIG. 104 , the anti-TF antibody affinity column canbind 7t15-TGFRs which contains TF as a fusion partner of 7t15-TGFRs. Thebuffer-exchanged protein sample was stored at 2-8° C. for furtherbiochemical analyses and biological activity tests. After each elution,the anti-TF antibody affinity column was stripped using 6 column volumesof 0.1M glycine (pH 2.5). The column was then neutralized using 5 columnvolumes of PBS, and 7 column volumes of 20% ethanol for storage. Theanti-TF antibody affinity column was connected to a GE Healthcare AKTAAvant system. The flow rate was 4 mL/min for all steps except for theelution step, which was 2 mL/min.

Reduced SDS-PAGE Analysis of 7t15-TGFRs

To determine the purity and molecular weight of the protein, 7t15-TGFRsprotein sample purified with anti-TF antibody affinity column wasanalyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPageBis-Tris gel) electrophoresis (SDS-PAGE) method under reduced condition.After electrophoresis, the gel was stained with InstantBlue for about 30min, followed by destaining overnight in purified water.

To verify that the 7t15-TGFRs protein undergoes glycosylation aftertranslation in CHO cells, a deglycosylation experiment was conductedusing the Protein Deglycosylation Mix II kit from New England Biolabsand the manufacturer's instructions. FIG. 105 shows reduced SDS-PAGEanalysis of the sample in non-deglycosylated (lane 1 in red outline) anddeglycosylated (lane 2 in yellow outline) state. These results showedthat the protein is glycosylated when it is expressed in CHO cells.After deglycosylation, the purified sample showed expected molecularweights (55 kDa and 39 kDa) in reduced SDS gel. Lane M was loaded with10 ul of SeeBlue Plus2 Prestained Standard.

Characterization of 7t15-TGFRs

7t15-TGFRs is a multi-chain polypeptide (a type A multi-chainpolypeptide described herein) that includes the first polypeptide thatis a soluble fusion of human IL-7, human tissue factor 219 fragment andhuman IL-15 (7t15), and the second polypeptide that is a soluble fusionof single chain two TGFβRII domains and sushi domain of human IL-15receptor alpha chain (TGFRs).

CHO cells were co-transfected with 7t15 and TGFRs vectors. The7t15-TGFRs complex was purified from the transfected CHO cell culturesupernatant. The IL-7, IL-15, TGFβ receptor and tissue factor (TF)components were demonstrated in the complex by ELISA as shown in FIG.106 . A humanized anti-TF monoclonal antibody (anti-TF IgG1) was used asthe capture antibody to determine TF in 7t15-TGFRs, and biotinylatedantibodies against human IL-15 antibody (R&D systems), human IL-7(Biolegend), anti-TGFβ receptor (R&D Systems) were used as the detectionantibodies to respectively determine IL-7, IL-15 and TGFβ receptor in7t15-TGFRs. Peroxidase conjugated streptavidin (Jackson ImmunoResearchLab) and ABTS substrate (Surmodics IVD, Inc.) were then used to detectthe bound biotinylated antibodies. The results were analyzed by ELISA(FIG. 106 ).

In Vivo Characterization of 7t15-TGFRs in C57BL/6 Mice

To determine the immunostimulatory activity of 7t15-TGFRs in vivo,C57BL/6 mice were subcutaneously treated with control solution (PBS) or7t15-TGFRs at 0.3, 1, 3 and 10 mg/kg. The treated mice were euthanized.The mouse spleens were collected and weighed day 4 post treatment.Single splenocyte suspensions were prepared and stained withfluorochrome-conjugated anti-CD4, anti-CD8, and anti-NK1.1 antibodiesand the percentage of CD4⁺ T cells, CD8⁺ T cells, and NK cells wasanalyzed by flow cytometry. The results showed that 7t15-TGFRs waseffective at expanding splenocytes based on spleen weight (FIG. 107A),especially at 1-10 mg/kg. The percentages of CD8⁺ T cells and NK cellswere higher compared to control-treated mice (FIG. 107B) at all dosestested.

CD44 Expression of CD4⁺ and CD8⁺ T Cells

It has been known that IL-15 induces CD44 expression on T cells anddevelopment of memory T cells. CD44 expression of CD4⁺ and CD8⁺ T cellsin the 7t15-TGFRs treated mice were assessed. C57BL/6 mice weresubcutaneously treated with 7t15-TGFRs. The splenocytes were stainedwith fluorochrome-conjugated anti-CD4, anti-CD8 and anti-CD44 monoclonalantibodies for immunocyte subsets. The percentages of CD4⁺CD44^(high) Tcells of total CD4+ T cells and CD8⁺CD44^(high) T cells of total CD8⁺ Tcells were analyzed by flow cytometry. As shown in FIGS. 108A and 108B,7t15-TGFRs significantly activated CD4⁺ and CD8⁺ T cells todifferentiate into memory T cells.

Furthermore, the dynamic proliferation of immune cells based on Ki67expression of splenocytes and cytotoxicity potential based on granzyme Bexpression of the splenocytes induced by 7t15-TGFRs after the singledose treatment of mouse were also evaluated. C57BL/6 mice weresubcutaneously treated with 7t15-TGFRs at 3 mg/kg. The treated mice wereeuthanized and the splenocytes were prepared. The prepared splenocyteswere stained with fluorochrome-conjugated anti-CD4, anti-CD8, andanti-NK1.1 (NK) antibodies for immunocyte subsets and thenintracellularly stained with anti-Ki67 antibody for cell proliferationand anti-granzyme B antibody for cytotoxic marker.

The mean fluorescent intensity (MFI) of Ki67 and granzyme B ofcorresponding immunocyte subsets was analyzed by flow cytometry. Asshown in FIGS. 109A and 109B, in the spleens of mice treated with7t15-TGFRs, the expression of Ki67 and granzyme B by CD8⁺ T cells and NKcells increased compared with PBS control treatment. These resultsdemonstrate that 7t15-TGFRs is not only to increase numbers of CD8⁺ Tcells and NK cells but also enhance potential cytotoxicity of thesecells.

Additionally, cytotoxicity of the mouse splenocytes against tumor cellswas also evaluated. Mouse Yac-1 cells were labeled with CELLTRACE®,violet dye, and used as tumor target cells. The splenocytes wereprepared from 7t15-TGFRs-treated mice and used as effector cells. Thetarget cells were mixed with effector cells at E:T ratio=10:1 in RPMI-10medium with or without 7t15-TGFRs at 100 nM and incubated at 37° C. for20 hours. Target Yac-1 cell inhibition was assessed by analysis ofviable violet-labeled Yac-1 cells using flow cytometry. Percentage ofYac-1 inhibition was calculated using a formula, (1−viable Yac-1 cellnumber in experimental sample/viable Yac-1 cell number in the samplewithout splenocytes)×100. As shown in FIG. 110 , 7t15-TGFRs-treatedmouse splenocytes had stronger cytotoxicity against Yac-1 cells than thecontrol mouse splenocytes and addition of 7t15-TGFRs during cytotoxicassay further enhanced cytotoxicity of splenocytes against Yac-1 targetcells.

Example 56: TGFRt15-21s137L Fusion Protein Generation andCharacterization

A fusion protein complex was generated comprising IL-21/IL-15RαSu/CD137Land TGFβ Receptor II/TF/IL-15 fusion proteins (FIG. 111 and FIG. 112 ).The human TGFβ Receptor II (Ile24-Asp159), tissue factor 219, and IL-15sequences were obtained from the UniProt website and DNA for thesesequences was synthesized by Genewiz. Specifically, a construct was madelinking two TGFβ Receptor II sequences with a G45(3) linker to generatea single chain version of TGFβ Receptor II and then directly linking tothe N-terminus coding region of tissue factor 219 followed by theN-terminus coding region of IL-15.

The nucleic acid sequence of the TGFRt15 construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC(Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanTissue Factor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRt15 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

The nucleic acid and protein sequences of the 21s137L are shown below.The nucleic acid sequence of the 21s137L construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC(Human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R α sushidomain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTC GGAA

The amino acid sequence of 21s137L fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIR((G4S)3 linker) GGGGSGGGGSGGGGS (Human CD137L)REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

The IL-21/IL-15RαSu/CD137L and TGFR/TF/IL-15 constructs were cloned intoa modified retrovirus expression vectors as described previously (HughesM S, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y, et al. Transfer ofa TCR gene derived from a patient with a marked antitumor responseconveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1cells. Co-expression of the two constructs in CHO-K1 cells allowed forformation and secretion of the soluble TGFR/TF/IL-15:IL-21/IL-15RαSu/CD137L protein complex (referred to as TGFRt15-21s137L),which can be purified by anti-TF IgG1 affinity and other chromatographymethods.

Purification Elution Chromatograph of TGFRt15-21s137L Using Anti-TFAntibody Affinity Column

TGFRt15-21s137L harvest from cell culture was loaded onto the anti-TFantibody affinity column equilibrated with 5 column volumes of PBS.After sample loading, the column was washed with 5 column volumes ofPBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0with 1M Tris base. The neutralized sample was then buffer exchanged intoPBS using Amicon centrifugal filters with a 30 KDa molecular weightcutoff. As shown in FIG. 113 , the anti-TF antibody affinity columnbound to TGFRt15-21s137L which contains TF as a fusion partner ofTGFRt15-21s137L. The buffer-exchanged protein sample was stored at 2-8°C. for further biochemical analyses and biological activity tests. Aftereach elution, the anti-TF antibody affinity column was stripped using 6column volumes of 0.1M glycine (pH 2.5). The column was then neutralizedusing 5 column volumes of PBS, and 7 column volumes of 20% ethanol forstorage. The anti-TF antibody affinity column was connected to a GEHealthcare AKTA Avant system. The flow rate was 4 mL/min for all stepsexcept for the elution step, which was 2 mL/min.

Example 57: TGFRt15-TGFRs21 Fusion Protein Generation andCharacterization

A fusion protein complex was generated comprising of TGFβ ReceptorII/IL-15RαSu/IL-21 and TGFβ Receptor II/TF/IL-15 fusion proteins (FIG.114 and FIG. 115 ). The human TGFβ Receptor II (Ile24-Asp159), tissuefactor 219, IL-21, and IL-15 sequences were obtained from the UniProtwebsite and DNA for these sequences was synthesized by Genewiz.Specifically, a construct was made linking two TGFβ Receptor IIsequences with a G45(3) linker to generate a single chain version ofTGFβ Receptor II and then directly linking to the N-terminus codingregion of tissue factor 219 followed by the N-terminus coding region ofIL-15.

The nucleic acid and protein sequences of a construct comprising twoTGFβ Receptor II linked to the N-terminus of tissue factor 219 followingwith the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the TGFRt15 construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC(Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanTissue Factor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRt15 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Constructs were also made by attaching two TGFβ Receptor II directly tothe IL-15RαSu chain, followed by the N-terminus coding region of IL-21,which was synthesized by Genewiz. The nucleic acid and protein sequencesof a construct comprising the TGFβ Receptor II linked to the N-terminusof IL-15RαSu following with the N-terminus of IL-21 are shown below.

The nucleic acid sequence of the TGFRs21 construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCT ACTCC(Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanIL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Human IL-21)CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC

The amino acid sequence of TGFRs21 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR(Human IL-21) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

The TGFR/IL-15RαSu/IL-21 and TGFR/TF/IL-15 constructs were cloned into amodified retrovirus expression vectors as described previously (Hughes MS, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y, et al. Transfer of aTCR gene derived from a patient with a marked antitumor response conveyshighly active T-cell effector functions. Hum Gene Ther 2005; 16:457-72),and the expression vectors were transfected into CHO-K1 cells.Co-expression of the two constructs in CHO-K1 cells allowed forformation and secretion of the solubleTGFR/TF/IL-15:TGFR/IL-15RαSu/IL-21 protein complex (referred to asTGFRt15-TGFRs21), which can be purified by anti-TF antibody IgG1affinity and other chromatography methods.

Purification Elution Chromatograph of TGFRt15-TGFRs21 Using Anti-TFAntibody Affinity Column

TGFRt15-TGFRs21 harvested from cell culture was loaded onto the anti-TFantibody affinity column equilibrated with 5 column volumes of PBS.After sample loading, the column was washed with 5 column volumes ofPBS, followed by elution with 6 column volumes of 0.1M acetic acid (pH2.9). A280 elution peak was collected and then neutralized to pH 7.5-8.0with 1M Tris base. The neutralized sample was then buffer exchanged intoPBS using Amicon centrifugal filters with a 30 KDa molecular weightcutoff. As shown in FIG. 116 , the anti-TF antibody affinity columnbound to TGFRt15-TGFRs21 which contains TF as a fusion partner. Thebuffer-exchanged protein sample was stored at 2-8° C. for furtherbiochemical analyses and biological activity tests. After each elution,the anti-TF antibody affinity column was stripped using 6 column volumesof 0.1M glycine (pH 2.5). The column was then neutralized using 5 columnvolumes of PBS, and 7 column volumes of 20% ethanol for storage. Theanti-TF antibody affinity column was connected to a GE Healthcare AKTAAvant system. The flow rate was 4 mL/min for all steps except for theelution step, which was 2 mL/min.

Reduced SDS-PAGE Analysis of TGFRt15-TGFRs21

To determine the purity and molecular weight of the protein,TGFRt15-TGFRs21 protein sample purified with anti-TF antibody affinitycolumn was analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12%NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method under reducedcondition. After electrophoresis, the gel was stained with InstantBluefor about 30 min, followed by destaining overnight in purified water.

To verify that the TGFRt15-TGFRs21 protein undergoes glycosylation aftertranslation in CHO cells, a deglycosylation experiment was conductedusing the Protein Deglycosylation Mix II kit from New England Biolabsand the manufacturer's instructions. FIG. 117 shows the reduced SDS-PAGEanalysis of the sample in non-deglycosylated (lane 1 in red outline) anddeglycosylated (lane 2 in yellow outline) state. It is clear that theprotein is glycosylated when it is expressed in CHO cells. Afterdeglycosylation, the purified sample showed expected molecular weights(69 kDa and 55 kDa) in reduced SDS gel. Lane M was loaded with 10 ul ofSeeBlue Plus2 Prestained Standard.

Immunostimulation of TGFRt15-TGFRs21 in C57BL/6 Mice

TGFRt15-TGFRs21 is a multi-chain polypeptide (a type A multi-chainpolypeptide described herein) that includes the first polypeptide thatis a soluble fusion of single chain two TGFβRII domains, human tissuefactor 219 fragment and human IL-15 (TGFRt15), and the secondpolypeptide that is a soluble fusion of single chain two TGFβRIIdomains, sushi domain of human IL-15 receptor alpha chain and humanIL-21 (TGFRs21).

CHO cells were co-transfected with TGFRt15 and TGFRs21 vectors. TheTGFRt15-TGFRs21 complex was purified from the transfected CHO cellculture supernatant. The TGFβ receptor, IL-15, IL-21 and tissue factor(TF) components were demonstrated in the complex by ELISA as shown inFIG. 118A-B. A humanized anti-TF monoclonal antibody (anti-TF IgG1) wasused as the capture antibody to determine TF in TGFRt15-TGFRs21,biotinylated anti-human IL-15 antibody (R&D systems), biotinylatedanti-human TGFβ receptor antibody (R&D systems, and biotinylatedanti-human IL-21 antibody (R&D Systems) were used as the detectionantibodies to respectively determine IL-15, TGFβ receptor, and IL-21 inTGFRt15-TGFRs21. For detection, peroxidase conjugated streptavidin(Jackson ImmunoResearch Lab) and ABTS were used.

Wild type C57BL/6 mice were treated subcutaneously with either controlsolution (PBS) or with TGFRt15-TGFRs21 at 3 mg/kg. Four days aftertreatment, spleen weight and the percentages of various immune celltypes present in the spleen were evaluated. As shown in FIG. 119A, thepercentages of CD4⁺ T cells, CD8⁺ T cells, and NK cells present in thespleen of control-treated and TGFRt15-TGFRs21-treated mice wereevaluated. The dynamic proliferation of immune cells based on Ki67expression after TGFRt15-TGFRs21 treatment was also evaluated. Thesplenocytes were stained with fluorochrome-conjugated anti-CD4,anti-CD8, and anti-NK1.1 (NK) antibodies and then intracellularlystained with anti-Ki67 antibody. The percentage of CD4⁺ T cells, CD8⁺ Tcells, and NK cells and the mean fluorescent intensity (MFI) of Ki67 ofcorresponding immunocyte subsets were analyzed by flow cytometry (FIGS.119A and 119B). Furthermore, cytotoxicity potential based on granzyme Bexpression of the splenocytes induced by TGFRt15-TGFRs21 after thesingle dose treatment of mouse was also evaluated. As shown in FIG. 120, in the spleens of mice treated with TGFRt15-TGFRs21, the expression ofgranzyme B by NK cells increased after treatment. The splenocytes fromTGFRt15-TGFRs21-treated mice were stained with fluorochrome-conjugatedanti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies and thenintracellularly stained with anti-granzyme B antibody. The meanfluorescent intensity (MFI) of granzyme B of corresponding immunocytesubsets was analyzed by flow cytometry (FIG. 120 ).

As shown in FIG. 119A, in the spleens of mice treated withTGFRt15-TGFRs21, the percentages of CD8⁺ T cells and NK cells bothincreased on day 4 after a single TGFRt15-TGFRs21 treatment. Theseresults demonstrate that TGFRt15-TGFRs21 is able to induce immune cellsto proliferate in mouse spleen, in particular CD8⁺ T cells and NK cells.

Additionally, cytotoxicity of the mouse splenocytes against tumor cellswas also evaluated. Mouse Yac-1 cells were labeled with CELLTRACE®,violet dye, and used as tumor target cells. The splenocytes wereprepared from TGFRt15-TGFRs21-treated mice and used as effector cells.The target cells were mixed with effector cells at E:T ratio=10:1 inRPMI-10 medium with or without TGFRt15-TGFRs21 at 100 nM and incubatedat 37° C. for 24 hours. Target Yac-1 cell inhibition was assessed byanalysis of viable violet-labeled Yac-1 cells using flow cytometry.Percentage of Yac-1 inhibition was calculated using a formula,(1−[viable Yac-1 cell number in experimental sample]/[viable Yac-1 cellnumber in the sample without splenocytes])×100. As shown in FIG. 121 ,TGFRt15-TGFRs21-treated mouse splenocytes had stronger cytotoxicityagainst Yac-1 cells than the control mouse cells in the presence ofTGFRt15-TGFRs21 during cytotoxic assay (FIG. 121 ).

Example 58: TGFRt15-TGFRs16 Fusion Protein Generation

A fusion protein complex was generated comprising of TGFβ ReceptorII/IL-15RαSu/anti-CD16scFv and TGFβ Receptor II/TF/IL-15 fusion proteins(FIG. 122 and FIG. 123 ). The human TGFβ Receptor II (Ile24-Asp159),tissue factor 219, and IL-15 sequences were obtained from the UniProtwebsite and DNA for these sequences was synthesized by Genewiz.Specifically, a construct was made linking two TGFβ Receptor IIsequences with a G45(3) linker to generate a single chain version ofTGFβ Receptor II and then directly linking to the N-terminus codingregion of tissue factor 219 followed by the N-terminus coding region ofIL-15.

The nucleic acid and protein sequences of a construct comprising twoTGFβ Receptor II linked to the N-terminus of tissue factor 219 followingwith the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the TGFRt15 construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanTissue Factor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRt15 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Constructs were also made by attaching two TGFβ Receptor II directly tothe IL-15RαSu chain, followed by the anti-CD16scFv sequence, which wassynthesized by Genewiz. The nucleic acid and protein sequences of aconstruct comprising the TGFβ Receptor II linked to the N-terminus ofIL-15RαSu following with the anti-CD16scFγ sequence are shown below.

The nucleic acid sequence of the TGFRs16 construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanIL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Anti-human CD16scFv)TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCA CCCTGGTGACCGTGTCCAGG

The amino acid sequence of TGFRs16 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR(Anti-human CD16scFv) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

The TGFR/IL-15RαSu/anti-CD16scFv and TGFR/TF/IL-15 constructs werecloned into a modified retrovirus expression vectors as describedpreviously (Hughes M S, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y,et al. Transfer of a TCR gene derived from a patient with a markedantitumor response conveys highly active T-cell effector functions. HumGene Ther 2005; 16:457-72), and the expression vectors were transfectedinto CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cellsallowed for formation and secretion of the solubleTGFR/TF/IL-15:TGFR/IL-15RαSu/anti-CD16scFv protein complex (referred toas TGFRt15-TGFRs16), which can be purified by anti-TF IgG1 affinity andother chromatography methods.

Example 59: The TGFRt15-TGFRs137L Fusion Protein Generation

A fusion protein complex was generated comprising of TGFβ ReceptorII/IL-15RαSu/CD137L and TGFβ Receptor II/TF/IL-15 fusion proteins (FIG.124 and FIG. 125 ). The human TGFβ Receptor II (Ile24-Asp159), tissuefactor 219, CD137L, and IL-15 sequences were obtained from the UniProtwebsite and DNA for these sequences was synthesized by Genewiz.Specifically, a construct was made linking two TGFβ Receptor IIsequences with a G4S(3) linker to generate a single chain version ofTGFβ Receptor II and then directly linking to the N-terminus codingregion of tissue factor 219 followed by the N-terminus coding region ofIL-15.

The nucleic acid and protein sequences of a construct comprising twoTGFβ Receptor II linked to the N-terminus of tissue factor 219 followingwith the N-terminus of IL-15 are shown below.

The nucleic acid sequence of the TGFRt15 construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanTissue Factor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of TGFRt15 fusion protein (including the leadersequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human Tissue Factor 219)SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

Constructs were also made by attaching two TGFβ Receptor II directly tothe IL-15RαSu chain, followed by a (G4S)3 linker and the CD137Lsequence, which was synthesized by Genewiz. The nucleic acid and proteinsequences of a construct comprising the TGFβ Receptor II linked to theN-terminus of IL-15RαSu following with a (G4S)3 linker and the CD137Lsequence are shown below.

The nucleic acid sequence of the TGFRs137L construct (including signalpeptide sequence) is as follows:

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human TGFβ Receptor II fragments)ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCA GCAACCCCGAC (HumanIL-15R α sushi domain)ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGG AA

The amino acid sequence of TGFRs137L fusion protein (including theleader sequence) is as follows:

(Signal peptide) MKWVTFISLLFLFSSAYS (Human TGFβ Receptor II)IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKAT NVAHWTTPSLKCIR((G4S)3 linker) GGGGSGGGGSGGGGS (Human CD137L)REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptideto generate the mature form that may be soluble or secreted.

The TGFR/IL-15RαSu/CD137L and TGFR/TF/IL-15 constructs were cloned intoa modified retrovirus expression vectors as described previously (HughesM S, Yu Y Y, Dudley M E, Zheng Z, Robbins P F, Li Y, et al. Transfer ofa TCR gene derived from a patient with a marked antitumor responseconveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1cells. Co-expression of the two constructs in CHO-K1 cells allowed forformation and secretion of the solubleTGFR/TF/IL-15:TGFR/IL-15RαSu/CD137L protein complex (referred to asTGFRt15-TGFRs137L), which can be purified by anti-TF IgG1 affinity andother chromatography methods.

Example 60: Stimulation of NK Cells In Vitro by Multi-Chain ChimericPolypeptide Constructs

A set of experiments was performed to assess changes in the surfacephenotype of lymphocyte populations after stimulation with 18t15-12s,18t15-12s16, and 7t15-21s. In these experiments, fresh human leukocyteswere obtained from the blood bank. Peripheral blood lymphocytes wereisolated with the Ficoll-PAQUE Plus (GE Healthcare) density gradientmedia. The cells were counted and resuspended at 0.2×10⁶/mL in a 96-wellflat-bottom plate in 0.2 mL of complete media (RPMI 1640 (Gibco)supplemented with 2 mM L-glutamine (Thermo Life Technologies),penicillin (Thermo Life Technologies), streptomycin (Thermo LifeTechnologies), and 10% FBS (Hyclone)). The cells were stimulated with:18t15-12s (100 nM); 18t15-12s16 (100 nM), a mixture of single cytokinesrhIL15 (50 ng/mL) (Miltenyi), rhIL18 (50 ng/mL) (Invivogen), and rhIL-12(10 ng/mL) (Peprotech); 7t15-21s (100 nM)+anti-TF antibody (50 nM);7t15-21s (100 nM); or anti-TF antibody (50 nM) at 37° C. and 5% CO₂ for16 hours. The next day, the cells were harvested and surface stained for30 minutes with antibodies specific for CD4 or CD8, CD62L, and CD69.After surface staining, cells were washed (1500 RPM for 5 minutes atroom temperature) in FACS buffer (1×PBS (Hyclone) with 0.5% BSA (EMDMillipore) and 0.001% sodium azide (Sigma)). After two washes, the cellswere analyzed by flow cytometry (Celesta-BD Bioscience). FIG. 126 showsthat overnight incubation of purified lymphocyte populations (CD4 andCD8 T cells) with 18t15-12s, 18t15-12s16, or 7t15-21s+anti-TF antibodyresulted in an increase in the percentage of CD8 and CD4 T cellsexpressing CD69. Additionally, incubation with 7t15-21s+anti-TF antibodyresulted in an increase in the percentage of CD8 and CD4 T cellsexpressing CD62L (FIG. 126 ).

A set of experiments was performed to determine the increase inphospho-STAT4 and phospho-STAT5 levels in NK cells after stimulationwith 18t15-12s. In these experiments, fresh human leukocytes wereobtained from the blood bank and CD56⁺ NK cells were isolated with theRosetteSep/human NK cell reagent (StemCell Technologies).

The purity of NK cells was >70% and confirmed by staining withCD56-BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 specific antibodies(BioLegend). The cells were counted and resuspended in 0.05×10⁶/mL in a96-well flat-bottom plate in 0.1 mL of complete media (RPMI 1640 (Gibco)supplemented with 2 mM L-glutamine (Thermo Life Technologies),penicillin (Thermo Life Technologies), streptomycin (Thermo LifeTechnologies), and 10% FBS (Hyclone)). The cells were stimulated withhIL-12 (10 ng/mL) (Biolegend) or hIL-15 (50 ng/mL) (NCI) (Singlecytokines), or 18t15-12s (100 nM) at 37° C. and 5% CO₂ for 90 minutes.Unstimulated NK cells (US) were used as a control. The cells wereharvested and fixed in paraformaldehyde (Sigma) to a final concentrationof 1.6%. Plates were incubated in the dark at room temperature for 10minutes. FACS buffer (1×PBS (Hyclone) with 0.5% BSA (EMD Millipore) and0.001% sodium azide (Sigma)) (100 μL) was added and cells weretransferred to 96-well “V” bottom plate. The cells were washed for 1500RPM for 5 minutes at room temperature. The cell pellet was mixed with100 μL chilled methanol by gently pipetting up and down, and cells wereincubated for 30 minutes at 4° C. The cells were mixed with 100 mL ofFACS buffer and washed for 1500 RPM for 5 minutes at room temperature.The cell pellets were mixed with 50 mL of FACS buffer containing 4 mL ofpSTAT4 (BD Bioscience) and pSTAT5 antibodies (BD Bioscience) followed byincubation for 30 minutes at room temperature in the dark. The cellswere mixed with 100 mL of FACS buffer and washed for 1500 RPM for 5minutes at room temperature. The cell pellets were mixed with 50 mL ofFACS buffer and cells were analyzed by flow cytometry (Celesta-BDBioscience). FIG. 127 shows that incubation of NK cells with 18t15-12sinduced an increase in pSTAT4 and pSTAT5 (plotted data, normalizedfold-change).

Example 61: Stimulation of NK Cells In Vivo by TGFRt15-TGFRs

A set of experiments was performed to determine the effect of theTGFRt15-TGFRs construct on immune stimulation in ApoE^(−/−) mice fedwith a Western diet. In these experiments, 6-week old femaleB6.129P2-ApoE^(tm1Unc)/J mice (Jackson Laboratory) were fed with aWestern diet containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5%casein, and 15% starch (TD88137, Envigo Laboratories). After 8-weeks ofthe Western diet, the mice were injected subcutaneously withTGFRt15-TGFRs at 3 mg/kg. Three days post treatment, mice were fastedfor 16 hours and then blood samples were collected through retro-orbitalvenous plexus puncture. The blood was mixed with 10 μL 0.5 M EDTA, and20 μL blood was taken for lymphocyte subsets analysis. The red bloodcells were lysed with ACK (0.15 M NH₄Cl, 1.0 mM KHCO₃, 0.1 mM Na₂EDTA,pH 7.4) and the lymphocytes were stained with anti-mouse CD8a andanti-mouse NK1.1 antibodies for 30 minutes at 4° C. in FACS stainingbuffer (1% BSA in PBS). The cells were washed once and analyzed with aBD FACS Celesta. For Treg staining, ACK treated blood lymphocytes werestained with anti-mouse CD4 and anti-mouse CD25 antibodies for 30minutes at 4° C. in FACS staining buffer. The cells were washed once andresuspended in fixation/permeabilization working solution and incubatedat room temperature for 60 minutes. The cells were washed once andresuspended in permeabilization buffer. The samples were centrifuged at300-400×g for 5 minutes at room temperature and the supernatant was thendiscarded. The cell pellet was resuspended in residual volume and thevolume adjusted to about 100 μL with 1× permeabilization buffer.Anti-Foxp3 antibody was added to the cells, and the cells were incubatedfor 30 minutes at room temperature. Permeabilization buffer (200 μL) wasadded to the cells, and the cells were centrifuged at 300-400×g for 5minutes at room temperature. The cells were resuspended in flowcytometry staining buffer and analyzed on a flow cytometer. FIGS.128A-128C show that treatment with TGFRt15-TGFRs increased thepercentage of NK cells and CD8⁺ T cells in ApoE^(−/−) mice fed withWestern diet.

Example 62: Induction of Proliferation of Immune Cells In Vivo

A set of experiments was performed to determine the effect of theTGFRt15-TGFRs construct on immune stimulation in C57BL/6 mice. In theseexperiments, C57BL/6 mice were subcutaneously treated with controlsolution (PBS) or TGFRt15-TGFRs at 0.1, 0.3, 1, 3, and 10 mg/kg. Thetreated mice were euthanized 4 days post-treatment. Spleen weight wasmeasured and splenocyte suspensions were prepared. The splenocytesuspensions were stained with conjugated anti-CD4, anti-CD8, andanti-NK1.1 (NK) antibodies. The cells were additionally stained forproliferation marker Ki67. FIG. 129A shows that spleen weight in micetreated with TGFRt15-TGFRs increased with increasing dosage ofTGFRt15-TGFRs. Additionally, spleen weight in mice treated with 1 mg/kg,3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs was higher as compared to micetreated with just the control solution. The percentages of CD8⁺ T cellsand NK cells both increased with increasing dosage of TGFRt15-TGFRs(FIG. 129B). Finally, TGFRt15-TGFRs significantly upregulated expressionof cell proliferation marker Ki67 in both CD8⁺ T cells and NK cells atall doses of TGFRt15-TGFRs tested (FIG. 129C). These results demonstratethat TGFRt15-TGFRs treatment induced proliferation of both CD8⁺ T cellsand NK cells in C57BL/6 mice.

A set of experiments was performed to determine the effect of theTGFRt15-TGFRs construct on immune stimulation in ApoE^(−/−) mice fedwith a Western diet. In these experiments, 6-week old femaleB6.129P2-ApoE^(tm1Unc)/J mice (Jackson Laboratory) were fed with aWestern diet containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5%casein, and 15% starch (TD88137, Envigo Laboratories). After 8-week ofthe Western diet, the mice were injected subcutaneously withTGFRt15-TGFRs at 3 mg/kg. Three days post-treatment, the mice werefasted for 16 hours and then blood samples were collected throughretro-orbital venous plexus puncture. The blood was mixed with 10 μL 0.5M EDTA and 20 μL blood was taken for lymphocyte subsets analysis. Thered blood cells were lysed with ACK (0.15 M NH₄Cl, 1.0 mM KHCO₃, 0.1 mMNa₂EDTA, pH 7.4) and the lymphocytes were stained with anti-mouse CD8aand anti-mouse NK1.1 antibodies for 30 minutes at 4° C. in FACS stainingbuffer (1% BSA in PBS). The cells were washed once and resuspended inFixation Buffer (BioLegend Cat #420801) for 20 minutes at roomtemperature. The cells were centrifuged at 350×g for 5 minutes, thefixed cells were resuspended in Intracellular Staining PermeabilizationWash Buffer (BioLegend Cat #421002) and then centrifuged at 350×g for 5minutes. The cells were then stained with anti-Ki67 antibody for 20minutes at RT. The cells were washed twice with Intracellular StainingPermeabilization Wash Buffer and centrifuged at 350×g for 5 minutes. Thecells were then resuspended in FACS staining buffer. Lymphocyte subsetswere analyzed with a BD FACS Celesta. As described in FIGS. 130A and130B, treatment of ApoE^(−/−) mice with TGFRt15-TGFRs inducedproliferation (Ki67-positive staining) in NK and CD8⁺ T cells.

Example 63: NK-Mediated Cytotoxicity Following Treatment withMulti-Chain Construct

A set of experiments was performed to determine if treatment of NK cellswith TGFRt15-TGFRs enhanced cytotoxicity of NK cells. In theseexperiments, Human Daudi B lymphoma cells were labeled with CELLTRACE®,violet dye (CTV), and used as tumor target cells. Mouse NK effectorcells were isolated with NK1.1-positive selection using a magnetic cellsorting method (Miltenyi Biotec) of C57BL/6 female mouse spleens 4 dayspost TGFRt15-TGFRs subcutaneous treatment at 3 mg/kg. Human NK effectorcells were isolated from peripheral blood mononuclear cells derived fromhuman blood buffy coats with the RosetteSep/human NK cell reagent(Stemcell Technologies). The target cells (Human Daudi B lymphoma cells)were mixed with effector cells (either mouse NK effector cells or humanNK effector cells) in the presence of 50 nM TGFRt15-TGFRs or in theabsence of TGFRt15-TGFRs (control) and incubated at 37° C. for 44 hoursfor mouse NK cells and for 20 hours for human NK cells. Target cell(Daudi) viability was assessed by analysis of propidium iodide-positive,CTV-labeled cells using flow cytometry. The percentage of Daudiinhibition was calculated using the formula (1−viable tumor cell numberin experimental sample/viable tumor cell number in the sample without NKcells)×100. FIG. 131 shows that mouse (FIG. 131A) and human (FIG. 131B)NK cells had significantly stronger cytotoxicity against Daudi B cellsfollowing NK cell activation with TGFRt15-TGFRs than in the absence ofTGFRt15-TGFRs activation.

A set of experiments was performed to determine antibody-dependentcellular cytotoxicity (ADCC) of mouse and human NK cells followingtreatment with TGFRt15-TGFRs. In these experiments, human Daudi Blymphoma cells were labeled with CELLTRACE®, violet dye (CTV), and usedas tumor target cells. Mouse NK effector cells were isolated withNK1.1-positive selection using a magnetic cell sorting method (MiltenyiBiotec) of C57BL/6 female mouse spleens 4 days post-TGFRt15-TGFRssubcutaneous treatment at 3 mg/kg. Human NK effector cells were isolatedfrom peripheral blood mononuclear cells derived from human blood buffycoats with the RosetteSep/human NK cell reagent (Stemcell Technologies).The target cells (Daudi B cells) were mixed with effector cells (eithermouse NK effector cells or human NK effector cells) in the presence ofanti-CD20 antibody (10 nM Rituximab, Genentech) and in the presence of50 nM TGFRt15-TGFRs, or in the absence of TGFRt15-TGFRs (control) andincubated at 37° C. for 44 hours for mouse NK cells and for 20 hours forhuman NK cells. The Daudi B cells express the CD20 targets for theanti-CD20 antibody. Target cell viability was assessed after incubationby analysis of propidium iodide-positive, CTV-labeled target cells usingflow cytometry. The percentage of Daudi inhibition was calculated usingthe formula (1-viable tumor cell number in experimental sample/viabletumor cell number in the sample without NK cells)×100. FIG. 132 showsthat mouse NK cells (FIG. 132A) and human NK cells (FIG. 132B) hadstronger ADCC activity against Daudi B cells following NK cellactivation with TGFRt15-TGFRs than in the absence of TGFRt15-TGFRsactivation.

Example 64: Treatment of Cancer

A set of experiments was performed to assess antitumor activity ofTGFRt15-TGFRs plus anti-TRP1 antibody (TA99) in combination withchemotherapy in a melanoma mouse model. In these experiments, C57BL/6mice were subcutaneously injected with 0.5×10⁶ B16F10 melanoma cells.The mice were treated with three doses of chemotherapy docetaxel (10mg/kg) (DTX) on day 1, day 4, and day 7, followed by treatment withsingle dose of combination immunotherapy TGFRt15-TGFRs (3mg/kg)+anti-TRP1 antibody TA99 (200 μg) on day 9. FIG. 133A shows aschematic of the treatment regimen. Tumor growth was monitored bycaliper measurement, and tumor volume was calculated using the formulaV=(L×W²)/2, where L is the largest tumor diameter and W is theperpendicular tumor diameter. FIG. 133B shows that treatment withDTX+TGFRt15-TGFRs+TA99 significantly reduced tumor growth compared tosaline control and DTX treatment groups (N=10, ****p<0.001, Multiple ttest analyses).

To assess immune cell subsets in the B16F10 tumor model, peripheralblood analysis was performed. In these experiments, C57BL/6 mice wereinjected with B16F10 cells and treated with DTX, DTX+TGFRt15-TGFRs+TA99,or saline. Blood was drawn from the submandibular vein of B16F10tumor-bearing mice on days 2, 5, and 8 post-immunotherapy for theDTX+TGFRt15-TGFRs+TA99 group and day 11 post-tumor injection for the DTXand saline groups. RBCs were lysed in ACK lysis buffer and thelymphocytes were washed and stained with anti-NK1.1, anti-CD8, andanti-CD4 antibodies. The cells were analyzed by flow cytometry(Celesta-BD Bioscience). FIGS. 133C-133E show thatDTX+TGFRt15-TGFRs+TA99 treatment induced an increase in the percentageof NK cells and CD8⁺ T cells in the tumors compared to the saline andDTX treatment groups.

On day 17, total RNA was extracted from tumors of mice treated withsaline, DTX or DTX+TGFRt15-TGFRs+TA99 using Trizol. Total RNA (1 μg) wasused for cDNA synthesis using the QUANTITECT® Reverse Transcription Kit(Qiagen). Real-time PCR was carried out with CFX96 Detection System(Bio-Rad) using FAM-labeled predesigned primers for senescence cellmarkers, (F) p21 (G) DPP4 and (H) IL6. The housekeeping gene 18Sribosomal RNA was used as an internal control to normalize thevariability in expression levels. The expression of each target mRNArelative to 18S rRNA was calculated based on Ct as 2-Δ(ΔCt), in whichΔCt=Cttarget−Ct18S. The data is presented as fold-change as compared tosaline control. FIG. 133F-133H show that DTX treatment induced anincrease in senescent tumor cells that were subsequently reducedfollowing treatment with TGFRt15−TGFRs+TA99 immunotherapy.

A set of experiments was performed to investigate amelioration ofWestern diet-induced hyperglycemia in ApoE^(−/−) mice by TGFRt15-TGFRs.In these experiments, 6-week old female B6.129P2-ApoE^(tm1Unc)/J mice(Jackson Laboratory) were fed with a Western diet containing 21% fat,0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137,Envigo Laboratories). After 8-weeks of the Western diet, the mice wereinjected subcutaneously with TGFRt15-TGFRs at 3 mg/kg. Three dayspost-treatment, the mice were fasted for 16 hours and then blood sampleswere collected through retro-orbital venous plexus puncture. Bloodglucose was detected with a glucose meter (OneTouch UltraMini) andGenUltimated test strips using a drop of fresh blood. As shown in FIG.134A, TGFRt15-TGFRs treatment reduced hyperglycemia induced by theWestern diet. The plasma insulin and resistin levels were analyzed withMouse Rat Metabolic Array by Eve Technologies. HOMA-IR was calculatedusing the following formula: homeostatic model assessment-insulinresistance=Glucose (mg/dL)*Insulin (mU/mL)/405. As shown in FIG. 134B,TGFRt15-TGFRs treatment reduced insulin resistance compared to theuntreated group. TGFRt15-TGFRs (p<0.05) reduced resistin levelssignificantly compared to the untreated group as shown in FIG. 142C,which may relate to the reduced insulin resistance induced byTGFRt15-TGFRs (FIG. 134B).

Example 65: Induction of Differentiation of NK Cells intoCytokine-Induced Memory Like NK Cells

A set of experiments was performed to assess the differentiation of NKcells into cytokine-induced memory like NK Cells (CIMK-NK Cells) afterstimulation with 18t15-12s. In these experiments, fresh human leukocyteswere obtained from the blood bank and CD56⁺ NK cells were isolated withthe RosetteSep/human NK cell reagent (StemCell Technologies). The purityof NK cells was >90% and confirmed by staining with CD56-BV421,CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). Thecells were counted and resuspended in 2×10⁶/mL in a 24-well flat-bottomplate in 2 mL of complete media (RPMI 1640 (Gibco) supplemented with 2mM L-glutamine (Thermo Life Technologies), penicillin (Thermo LifeTechnologies), streptomycin (Thermo Life Technologies), and 10% FBS(Hyclone)). The cells were unstimulated (“No Spike”) or stimulated with18t15-12s (100 nM) or a mixture of single cytokines including rhIL15 (50ng/mL) (Miltenyi), rhIL18 (50 ng/mL) (Invivogen), and rhIL-12 (10 ng/mL)(Peprotech) (“single cytokines”) at 37° C. and 5% CO₂ for 16 hrs. Thenext day, the cells were harvested, and washed two times with warmcomplete media at 1000 RPM for 10 minutes at room temperature. The cellswere resuspended at 2×10⁶/mL in a 24-well flat-bottom plate in 2 mL ofcomplete media with rhIL15 (1 ng/mL). After every 2 days, half of themedium was replaced with fresh complete media containing rhIL15.

To assess the change in memory phenotype of NK cells at day 7, the cellswere stained with antibodies to cell-surface CD56, CD16, CD27, CD62L,NKp30, and NKp44 (BioLegend). After surface staining, the cells werewashed (1500 RPM for 5 minutes at room temperature) in FACS buffer(1×PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide(Sigma)). After two washes, the cells were analyzed by flow cytometry(Celesta-BD Bioscience). FIG. 135 shows that incubation of NK cells with18t15-12s resulted in an increase in the percentage of CD16⁺CD56⁺ NKcells expressing CD27, CD62L, and NKp44, and an increase in the levels(MFI) of NKp30 in CD16⁺CD56⁺ NK cells.

Example 66: Upregulation of CD44 Memory T Cells

A set of experiments was performed to assess upregulation of CD44 memoryT cells upon treatment with TGFRt15-TGFRs. In these experiments, C57BL/6mice were subcutaneously treated with TGFRt15-TGFRs. The treated micewere euthanized and the single splenocyte suspensions were prepared 4days (TGFRt15-TGFRs) following the treatment. The prepared splenocyteswere stained with fluorochrome-conjugated anti-CD4, anti-CD8 andanti-CD44 antibodies and the percentages of CD44^(high) T cells in CD4⁺T cells or CD8⁺ T cells were analyzed by flow cytometry. The resultsshow that TGFRt15-TGFRs upregulated expression of the memory marker CD44on CD4⁺ and CD8⁺ T cells (FIG. 136 ). These findings indicate thatTGFRt15-TGFRs was able to induce mouse T cells to differentiate intomemory T cells.

Example 67: Tissue Factor Coagulation Assays Following Treatment withSingle-Chain or Multi-Chain Chimeric Polypeptides

A set of experiments was performed to assess blood coagulation followingtreatment with single-chain or multi-chain chimeric polypeptides. Toinitiate the blood coagulation cascade pathway, tissue factor (TF) bindsto Factor VIIa (FVIIa) to form a TF/FVIIa complex. The TF/FVIIa complexthen binds Factor X (FX) and converts FX to FXa.

Factor VIIa (FVIIa) Activity Assay

One assay to measure blood coagulation involves measuring Factor VIIa(FVIIa) activity. This type of assay requires the presence of tissuefactor and calcium. The TF/FVIIa complex activity can be measured by asmall substrate or by a natural protein substrate, for example, Factor X(FX). When FX is used as a substrate, phospholipids are also requiredfor TF/FVIIa activity. In this assay, FVIIa activity is determined withFVIIa-specific chromogenic substrate S-2288 (Diapharma, West Chester,Ohio). The color change of the S-2288 substrate can be measuredspectrophotometrically and is proportional to the proteolytic activityof FVIIa (e.g., the TF/FVIIa complex).

In these experiments, the FVIIa activity of the following groups werecompared: the 219-amino acid extracellular domain of tissue factordomain (TF₂₁₉), a multi-chain chimeric polypeptide with a wild-typetissue factor domain, and a multi-chain chimeric polypeptide with amutant tissue factor domain. The chimeric polypeptides containing mutanttissue factor molecules were constructed with mutations to the TF domainat amino acid sites: Lys20, Ile22, Asp58, Arg135, and Phe140.

In order to assess activity of FVIIa, FVIIa, and TF₂₁₉ or aTF₂₁₉-containing multi-chain chimeric polypeptide were mixed at an equalmolar concentration (10 nM) in all wells of a 96-well ELISA plate in atotal volume of 70 μL. After incubation for 10 minutes at 37° C., 10 μLof 8 mM S-2288 substrate was added to start the reaction. The incubationwas then kept at 37° C. for 20 minutes. Finally, color change wasmonitored by reading absorbance at 405 nm. The OD values of differentTF/VIIa complexes are shown in Table 1 and Table 2. Table 1 shows acomparison of TF₂₁₉, 21t15-215 wild-type (WT) and 21t15-21s mutant(Mut). Table 2 shows a comparison of TF₂₁₉, 21t15-TGFRs wild-type (WT),and 21t15-TGFRs mutant (Mut). These data show that TF₂₁₉-containingmulti-chain chimeric polypeptides (e.g., 21t15-21s-WT, 21t15-21s-Mut,21t15-TGFRS-WT, and 21t15-TGFRS-Mut) have lower FVIIa activity thanTF₂₁₉ when the chromogenic S-2288 was used as a substrate. Notably, themulti-chain chimeric polypeptides containing TF₂₁₉ mutations showed muchlower FVIIa activity when compared to multi-chain chimeric polypeptidescontaining wild type TF₂₁₉.

TABLE 1 FVIIa activity Molecule OD value at 405 nm TF₂₁₉ 0.30721t15/21S-WT 0.136 21t15/21S-Mut 0.095 WT: wild type of TF₂₁₉, Mut:TF₂₁₉ containing mutations.

TABLE 2 FVIIa activity Molecule OD value at 405 nm TF₂₁₉ 0.34521t15/TGFRS-WT 0.227 21t15/TGFRS-Mut 0.100 WT: wild type of TF₂₁₉, Mut:TF₂₁₉ containing mutations.Factor X (FX) Activation Assay

An additional assay to measure blood coagulation involves measuringactivation of Factor X (FX). Briefly, TF/VIIa activates bloodcoagulation Factor X (FX) to Factor Xa (FXa) in the presence of calciumand phospholipids. TF₂₄₃, which contains the transmembrane domain of TF,has much higher activity in activating FX to FXa than TF₂₁₉, which doesnot contain the transmembrane domain. TF/VIIa dependent activation of FXis determined by measuring FXa activity using an FXa-specificchromogenic substrate S-2765 (Diapharma, West Chester, Ohio). The colorchange of S-2765 can be monitored spectrophotometrically and isproportional to the proteolytic activity of FXa.

In these experiments, FX activation with a multi-chain chimericpolypeptide (18t15-12s, mouse (m)21t15, 21t15-TGFRs, and 21t15-7s) wascompared with a positive control (Innovin) or TF₂₁₉. TF₂₁₉ (orTF₂₁₉-containing multi-chain chimeric polypeptides)/FVIIa complexes weremixed at an equal molar concentration (0.1 nM each) in a volume of 50 μLin round bottom wells of a 96-well ELISA plate, after which 10 μL of 180nM FX was added. After 15 minutes of incubation at 37° C., during whichtime FX was converted to FXa, 8 μL of 0.5 M EDTA (which chelates calciumand thus terminates FX activation by TF/VIIa) was added to each well tostop FX activation. Next, 10 μL of 3.2 mM S-2765 substrate was added tothe reaction mixture. Immediately, the plate absorbance was measured at405 nm and was recorded as the absorbance at time 0. The plate was thenincubated for 10-20 minutes at 37° C. The color change was monitored byreading absorbance at 405 nm following the incubation. Results of FXactivation as measured by FXa activity using chromogenic substrateS-2765 are shown in FIG. 137 . In this experiment, Innovin, which is acommercial prothrombin reagent containing lipidated recombinant humanTF₂₄₃, was used as a positive control for FX activation. Innovin wasreconstituted with purified water to about 10 nM of TF₂₄₃. Next, 0.1 nMTF/VIIa complex was made by mixing an equal volume of 0.2 nM of FVIIawith 0.2 nM of Innovin. Innovin demonstrated very potent FX activationactivity, while TF₂₁₉ and TF₂₁₉-containing multi-chain chimericpolypeptides had very low FX activation activity, confirming that TF₂₁₉is not active in a TF/FVIIa complex for activating natural substrate FXin vivo.

Prothrombin Time Test

A third assay to measure blood coagulation is the prothrombin time (PT)test, which measures blood clotting activity. Here, the PT test wasperformed using commercially available normal human plasma (Ci-TrolCoagulation Control, Level I). For a standard PT test, clot reactionswere initiated by addition of Innovin, a lipidated recombinant humanTF₂₄₃, in the presence of calcium. Clotting time was monitored andreported by STart PT analyzer (Diagnostica Stago, Parsippany, N.J.). PTassays were started by injecting 0.2 mL of various dilutions of Innovindiluted in PT assay buffer (50 mM Tris-HCl, pH 7.5, 14.6 mM CaCl₂), 0.1%BSA) into cuvettes containing 0.1 mL of normal human plasma prewarmed at37° C. In the PT assay, shorter PT time (clotting time) indicates ahigher TF-dependent clotting activity while longer PT (clotting time)means lower TF-dependent clotting activity.

As seen in FIG. 138 , addition of different amounts of Innovin (e.g.,Innovin reconstituted with purified water equivalent to 10 nM oflipidated recombinant human TF₂₄₃ was considered to be 100% Innovin) tothe PT assay demonstrated a dose-response relationship, where lowerconcentrations of TF₂₄₃ resulted in a longer PT time (lower clottingactivity). For example, 0.001% Innovin had a PT time greater than 110seconds, which was almost the same as buffer alone.

In another experiment, the PT test was conducted on TF₂₁₉ andmulti-chain chimeric polypeptides including: 18t15-12s, 7t15-21s,21t15-TGFRs-WT, and 21t15-TGFRs-Mut. FIG. 139 show that TF₂₁₉ andTF₂₁₉-containing multi-chain chimeric polypeptides (at a concentrationof 100 nM) had prolonged PT times indicating extremely low or noclotting activity.

Studies were also conducted to evaluate whether incubating themulti-chain chimeric polypeptides in the presence of other cellscarrying receptors for the cytokine components of the multi-chainchimeric polypeptide (32Dβ or human PBMCs) would affect the clottingtime in the PT assay. To examine whether cells that express IL-15receptor (32Dβ cells) or IL-15 and IL-21 receptors (PBMCs) would bindIL-15-containing multi-chain chimeric polypeptides to mimic natural TFas a cellular FVIIa receptor, TF₂₁₉-containing multi-chain chimericpolypeptides (at a concentration of 100 nM for each molecule) werediluted in the PT assay buffer and preincubated with 32Dβ cells (at2×10⁵ cells/mL) or PBMC (at 1×10⁵ cells/mL) for 20-30 minutes at roomtemperature. The PT assay was then conducted as described above. FIGS.140 and 141 shows that TF₂₁₉ and TF₂₁₉-containing multi-chain chimericpolypeptides mixed with 32Dβ cells (FIG. 140 ) or PBMC (FIG. 141 ) at afinal concentration of 100 nM had prolonged PT times similar to0.001-0.01% Innovin (equivalent to 0.1 pM to 1.0 pM of TF₂₄₃). Expressedin percentage of relative TF₂₄₃ activity, TF₂₁₉-containing multi-chainchimeric polypeptides had 100,000 to 1,000,000 times lower TF dependentclotting activity when compared to Innovin. This demonstrated thatTF₂₁₉-containing multi-chain chimeric polypeptides had extremely low orno TF-dependent clotting activity, even while the molecules were boundto an intact cell membrane surface, such as 32Dβ or PBMCs.

Example 68: Characterization of 7t15-21s137L (Long Version)

The nucleic acid sequence of the 7t15 construct (including signalpeptide sequence) is as follows (SEQ ID NO: 107):

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL7) GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAG GAGCAT (Human TissueFactor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTT CCGGGAG (Human IL-15)AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of 7t15 fusion protein (including the leadersequence) is as follows (SEQ ID NO: 106):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL7)DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTK EH (Human TissueFactor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNR KSTDSPVECMGQEKGEFRE(Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS

The nucleic acid sequence of the 21s137L construct (including signalpeptide sequence) is as follows (SEQ ID NO: 225):

(Signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTA CTCC(Human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human IL-15R α sushidomain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG ((G4S)3 linker)GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (Human CD137L)CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGG AA

The amino acid sequence of 21s137L fusion protein (including the leadersequence) is as follows (SEQ ID NO: 226):

(Signal peptide) MKWVTFISLLFLFSSAYS (Human IL-21)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (Human IL-15R α sushi domain)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKA TNVAHWTTPSLKCIR((G4S)3 linker) GGGGSGGGGSGGGGS (Human CD137L)REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

The following experiment was conducted to evaluate whether the CD137Lportion in 7t15-21s137L was intact to bind to CD137 (4.1BB). On day 1, a96-well plate was coated with 100 μL (2.5 μg/mL) of GAH IgG Fc (G-102-C,R&D Systems) in R5 (coating buffer), overnight. On day 2, the plateswere washed three times and blocked with 300 μL of 1% BSA in PBS at 37°C. for 2 hrs. 10 ng/ml of 4.1BB/Fc (838-4B, R&D Systems) was added at100 μl/well for 2 hrs at room temperature. Following three washes,7t15-21s137L (long version) or 7t15-21s137Ls (short version) was addedstarting at 10 nM, or recombinant human 4.1BBL starting at 180 ng/mL,with 1/3 dilution, followed by incubation at 4° C. overnight. On day 3,the plates were washed three times, and 500 ng/mL of biotinylate-goatanti-human 4.1BBL (BAF2295, R&D Systems) was applied at 100 μL per well,followed by incubation at RT for 2 hrs. The plates were washed threetimes, and incubated with 0.25 μg/mL of HRP-SA (Jackson ImmuneResearch)at 100 μL per well for 30 min. The plates were then washed three times,and incubated with 100 μL of ABTS for 2 mins at RT. The results wereread at 405 nm. As shown in FIG. 142 , both 7t15-21s137L (long version)and 7t15-21s137L (short version) could interact with 4.1BB/Fc (darkdiamond and gray square) compared to the recombinant human 4.1BB ligand(rhCD137L, light gray star). 7t15-21s137L (long version) (dark diamond)interacted better with 4.1BB/Fc as compared to 7t15-21s137L (shortversion) (gray square).

The following experiments were conducted to evaluate whether thecomponents IL7, IL21, IL15, and 4.1BBL in 7t15-21s137L (long version)were intact to be detected by the individual antibody using ELISA. A96-well plate was coated with 100 μL (4 μg/mL) of anti-TF (human IgG1)in R5 (coating buffer) and incubated at RT for 2 hrs. The plates werewashed three times, and blocked with 100 μL of 1% BSA in PBS. Purified7t15-21s137L (long version) was added starting at 10 nM, and at 1/3dilution, followed by incubation at RT for 60 min. The plates werewashed three times, and 500 ng/mL of biotinylate-anti-IL7 (506602, R&DSystems), 500 ng/mL of biotinylate-anti-IL21 (13-7218-81, R&D Systems),50 ng/mL of biotinylate-anti-IL15 (BAM247, R&D Systems), or 500 ng/ml ofbiotinylate-goat anti-human 4.1BBL (BAF2295, R&D Systems) was added perwell and incubated at room temperature for 60 min. The plates werewashed three times and incubated with 0.25 μg/mL of HRP-SA (JacksonImmunoResearch) at 100 μL per well for 30 min at RT. The plates werewashed four times, and incubated with 100 μL of ABTS for 2 mins at roomtemperature. The absorbance results were read at 405 nm. As shown inFIG. 143A-143D, the components including IL7, IL21, IL15, and 4.1BBL in7t15-21s137L (long version) were detected by the individual antibodies.

The following experiment was conducted to evaluate the activity of IL15in 7t15-21s137L (long version) and 7t15-21s137L (short version). Theability of 7t15-21s137L (long version) and 7t15-21s137L (short version)to promote proliferation of IL2Rαβγ-expressing CTLL2 cells was comparedwith that of recombinant IL15. IL15 dependent CTLL2 cells were washedfive times with IMDM-10% FBS and seeded to the wells at 2×10⁴cells/well. Serially diluted 7t15-21s137L (long version), 7t15-21s137L(short version), or IL15 were added to the cells. Cells were incubatedin a CO₂ incubator at 37° C. for 3 days. Cell proliferation was detectedby adding 20 μL of PrestoBlue (A13261, ThermoFisher) to each well on day3 and incubated for an additional 4 hours in a CO₂ incubator at 37° C.Raw absorbance at 570-610 nm was read in a micro-titer plate reader. Asshown in FIG. 144 , 7t15-21s137L (long version), 7t15-21s137L (shortversion), and IL15 all promoted CTLL2 cell proliferation. The EC₅₀ of7t15-21s137L (long version), 7t15-21s137L (short version), and IL15 is51.19 pM, 55.75 pM, and 4.947 pM, respectively.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

EXEMPLARY EMBODIMENTS Embodiment A1

A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

(i) a first target-binding domain;

(ii) a soluble tissue factor domain; and

(iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

-   -   (i) a second domain of a pair of affinity domains; and    -   (ii) a second target-binding domain,

wherein the first chimeric polypeptide and the second chimericpolypeptide associate through the binding of the first domain and thesecond domain of the pair of affinity domains.

Embodiment A2

The multi-chain chimeric polypeptide of embodiment A1, wherein the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide.

Embodiment A3

The multi-chain chimeric polypeptide of embodiment A1, wherein the firstchimeric polypeptide further comprises a linker sequence between thefirst target-binding domain and the soluble tissue factor domain in thefirst chimeric polypeptide.

Embodiment A4

The multi-chain chimeric polypeptide of any one of embodiments A1-A3,wherein the soluble tissue factor domain and the first domain of thepair of affinity domains directly abut each other in the first chimericpolypeptide.

Embodiment A5

The multi-chain chimeric polypeptide of any one of embodiments A1-A3,wherein the first chimeric polypeptide further comprises a linkersequence between the soluble tissue factor domain and the first domainof the pair of affinity domains in the first chimeric polypeptide.

Embodiment A6

The multi-chain chimeric polypeptide of any one of embodiments A1-A5,wherein the second domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide.

Embodiment A7

The multi-chain chimeric polypeptide of any one of embodiments A1-A5,wherein second chimeric polypeptide further comprises a linker sequencebetween the second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

Embodiment A8

The multi-chain chimeric polypeptide of any one of embodiments A1-A7,wherein the first target-binding domain and the second target-bindingdomain bind specifically to the same antigen.

Embodiment A9

The multi-chain chimeric polypeptide of embodiment A8, wherein the firsttarget-binding domain and the second target-binding domain bindspecifically to the same epitope.

Embodiment A10

The multi-chain chimeric polypeptide of embodiment A9, wherein the firsttarget-binding domain and the second target-binding domain comprise thesame amino acid sequence.

Embodiment A11

The multi-chain chimeric polypeptide of any one of embodiments A1-A7,wherein the first target-binding domain and the second target-bindingdomain bind specifically to different antigens.

Embodiment A12

The multi-chain chimeric polypeptide of any one of embodiments A1-A11,wherein one or both of the first target-binding domain and the secondtarget-binding domain is an antigen-binding domain.

Embodiment A13

The multi-chain chimeric polypeptide of embodiment A12, wherein thefirst target-binding domain and the second target-binding domain areeach antigen-binding domains.

Embodiment A14

The multi-chain chimeric polypeptide of embodiment A12 or A13, whereinantigen-binding domain comprises a scFv or a single domain antibody.

Embodiment A15

The multi-chain chimeric polypeptide of any one of embodiments A1-A14,wherein one or both of the first target-binding domain and the secondtarget-binding domain bind specifically to a target selected from thegroup consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123,IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4,MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R,MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3,EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-binding protein, HLA-DR, DLL4,TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligand of TGF-β receptor II(TGF-βRII), a ligand of TGF-βRIII, a ligand of DNAM-1, a ligand ofNKp46, a ligand of NKp44, a ligand of NKG2D, a ligand of NKP30, a ligandfor a scMHCI, a ligand for a scMHCII, a ligand for a scTCR, a receptorfor IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7,a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, areceptor for IL-15, a receptor for IL-17, a receptor for IL-18, areceptor for IL-21, a receptor for PDGF-DD, a receptor for stem cellfactor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand(FLT3L), a receptor for MICA, a receptor for MICB, a receptor for aULP16-binding protein, a receptor for CD155, a receptor for CD122, and areceptor for CD28.

Embodiment A16

The multi-chain chimeric polypeptide of any one of embodiments A1-A14,wherein one or both of the first target-binding domain and the secondtarget-binding domain is a soluble interleukin or cytokine protein.

Embodiment A17

The multi-chain chimeric polypeptide of embodiment A16, wherein thesoluble interleukin, cytokine, or ligand protein is selected from thegroup consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment A18

The multi-chain chimeric polypeptide of any one of embodiments A1-A14,wherein one or both of the first target-binding domain and the secondtarget-binding domain is a soluble interleukin or cytokine receptor.

Embodiment A19

The multi-chain chimeric polypeptide of embodiment A18, wherein thesoluble receptor is a soluble TGF-β receptor II (TGF-β RII), a solubleTGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a solubleNKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155,or a soluble CD28.

Embodiment A20

The multi-chain chimeric polypeptide of any one of embodiments A1-A19,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domain(s), where at least one of the one ormore additional antigen-binding domain(s) is positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains.

Embodiment A21

The multi-chain chimeric polypeptide of embodiment A20, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe soluble tissue factor domain and the at least one of the one or moreadditional antigen-binding domain(s), and/or a linker sequence betweenthe at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment A22

The multi-chain chimeric polypeptide of any one of embodiments A1-A19,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domains at the N-terminal and/or C-terminalend of the first chimeric polypeptide.

Embodiment A23

The multi-chain chimeric polypeptide of embodiment A22, wherein at leastone of the one or more additional target-binding domains directly abutsthe first domain of the pair of affinity domains in the first chimericpolypeptide.

Embodiment A24

The multi-chain chimeric polypeptide of embodiment A22, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first domain of the pair of affinity domains.

Embodiment A25

The multi-chain chimeric polypeptide of embodiment A22, wherein the atleast one of the one or more additional target-binding domains directlyabuts the first target-binding domain in the first chimeric polypeptide.

Embodiment A26

The multi-chain chimeric polypeptide of embodiment A22, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first target-binding domain.

Embodiment A27

The multi-chain chimeric polypeptide of embodiment A22, wherein at leastone of the one or more additional target-binding domains is disposed atthe N- and/or C-terminus of the first chimeric polypeptide, and at leastone of the one or more additional target-binding domains is positionedbetween the soluble tissue factor domain and the first domain of thepair of affinity domains in the first chimeric polypeptide.

Embodiment A28

The multi-chain chimeric polypeptide of embodiment A27, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the N-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment A29

The multi-chain chimeric polypeptide of embodiment A27, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment A30

The multi-chain chimeric polypeptide of embodiment A27, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the C-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment A31

The multi-chain chimeric polypeptide of embodiment A27, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment A32

The multi-chain chimeric polypeptide of embodiment A27, wherein the atleast one of the one or more additional target-binding domainspositioned between the soluble tissue factor domain and the first domainof the pair of affinity domains, directly abuts the soluble tissuefactor domain and/or the first domain of the pair of affinity domains.

Embodiment A33

The multi-chain chimeric polypeptide of embodiment A27, wherein thefirst chimeric polypeptide further comprises a linker sequence disposed(i) between the soluble tissue factor domain and the at least one of theone or more additional target-binding domains positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains, and/or (ii) between the first domain of the pair ofaffinity domains and the at least one of the one or more additionaltarget-binding domains positioned between the soluble tissue factordomain and the first domain of the pair of affinity domains.

Embodiment A34

The multi-chain chimeric polypeptide of any one of embodiments A1-A33,wherein the second chimeric polypeptide further comprises one or moreadditional target-binding domains at the N-terminal end or theC-terminal end of the second chimeric polypeptide.

Embodiment A35

The multi-chain chimeric polypeptide of embodiment A34, wherein at leastone of the one or more additional target-binding domains directly abutsthe second domain of the pair of affinity domains in the second chimericpolypeptide.

Embodiment A36

The multi-chain chimeric polypeptide of embodiment A34, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second domain of the pair of affinity domains in the second chimericpolypeptide.

Embodiment A37

The multi-chain chimeric polypeptide of embodiment A34, wherein at leastone of the one or more additional target-binding domains directly abutsthe second target-binding domain in the second chimeric polypeptide.

Embodiment A38

The multi-chain chimeric polypeptide of embodiment A34, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second target-binding domain in the second chimeric polypeptide.

Embodiment A39

The multi-chain chimeric polypeptide of any one of embodiments A20-A38,wherein two or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains bind specifically to the same antigen.

Embodiment A40

The multi-chain chimeric polypeptide of embodiment A39, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same epitope.

Embodiment A41

The multi-chain chimeric polypeptide of embodiment A40, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains comprisethe same amino acid sequence.

Embodiment A42

The multi-chain chimeric polypeptide of embodiment A39, wherein thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains each bind specifically tothe same antigen.

Embodiment A43

The multi-chain chimeric polypeptide of embodiment A42, wherein thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains each bind specifically tothe same epitope.

Embodiment A44

The multi-chain chimeric polypeptide of embodiment A43, wherein thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains each comprise the sameamino acid sequence.

Embodiment A45

The multi-chain chimeric polypeptide of any one of embodiments A20-A38,wherein the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to different antigens.

Embodiment A46

The multi-chain chimeric polypeptide of any one of embodiments A20-A45,wherein one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more target-binding domains is anantigen-binding domain.

Embodiment A47

The multi-chain chimeric polypeptide of embodiment A46, wherein thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains are each anantigen-binding domain.

Embodiment A48

The multi-chain chimeric polypeptide of embodiment A47, whereinantigen-binding domain comprises a scFv.

Embodiment A49

The multi-chain chimeric polypeptide of any one of embodiments A20-A48,wherein one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more target-binding domains bindspecifically to a target selected from the group consisting of: CD16a,CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R,IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand ofTGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, aligand of NKG2D, a ligand of NKP30, a ligand for a scMHCI, a ligand fora scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor forIL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, a receptor for CD122, and a receptor for CD3, and areceptor for CD28.

Embodiment A50

The multi-chain chimeric polypeptide of any one of embodiments A20-A48,wherein one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains is a soluble interleukin or cytokine protein.

Embodiment A51

The multi-chain chimeric polypeptide of embodiment A50, wherein thesoluble interleukin, cytokine, or ligand protein is selected from thegroup consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment A52

The multi-chain chimeric polypeptide of any one of embodiments A20-A48,wherein one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains is a soluble interleukin or cytokine receptor.

Embodiment A53

The multi-chain chimeric polypeptide of embodiment A52, wherein thesoluble receptor is a soluble TGF-β receptor II (TGF-β RII), a solubleTGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a solubleNKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155,a soluble CD122, a soluble CD3, or a soluble CD28.

Embodiment A54

The multi-chain chimeric polypeptide of any one of embodiments A1-A53,wherein the first chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the first chimericpolypeptide.

Embodiment A55

The multi-chain chimeric polypeptide of any one of embodiments A1-A53,wherein the second chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the second chimericpolypeptide.

Embodiment A56

The multi-chain chimeric polypeptide of any one of embodiments A1-A55,wherein the soluble tissue factor domain is a soluble human tissuefactor domain.

Embodiment A57

The multi-chain chimeric polypeptide of embodiment A56, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 1.

Embodiment A58

The multi-chain chimeric polypeptide of embodiment A57, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 1.

Embodiment A59

The multi-chain chimeric polypeptide of embodiment A58, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 1.

Embodiment A60

The multi-chain chimeric polypeptide of any one of embodiments A56-A59,wherein the soluble human tissue factor domain does not comprise one ormore of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment A61

The multi-chain chimeric polypeptide of embodiment A60, wherein thesoluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment A62

The multi-chain chimeric polypeptide of any one of embodiments A1-A61,wherein the soluble tissue factor domain is not capable of binding toFactor VIIa.

Embodiment A63

The multi-chain chimeric polypeptide of any one of embodiments A1-A62,wherein the soluble tissue factor domain does not convert inactiveFactor X into Factor Xa.

Embodiment A64

The multi-chain chimeric polypeptide of any one of embodiments A1-A63,wherein the multi-chain chimeric polypeptide does not stimulate bloodcoagulation in a mammal.

Embodiment A65

The multi-chain chimeric polypeptide of any one of embodiments A1-A64,wherein the pair of affinity domains is a sushi domain from an alphachain of human IL-15 receptor (IL15Rα) and a soluble IL-15.

Embodiment A66

The multi-chain chimeric polypeptide of embodiment A65, wherein thesoluble IL15 has a D8N or D8A amino acid substitution.

Embodiment A67

The multi-chain chimeric polypeptide of embodiment A65 or A66, whereinthe human IL15Rα is a mature full-length IL15Rα.

Embodiment A68

The multi-chain chimeric polypeptide of any one of embodiments A1-A64,wherein the pair of affinity domains is selected from the groupconsisting of: barnase and barnstar, a PKA and an AKAP, adapter/dockingtag modules based on mutated RNase I fragments, and SNARE modules basedon interactions of the proteins syntaxin, synaptotagmin, synaptobrevin,and SNAP25.

Embodiment A69

The multi-chain chimeric polypeptide of any one of embodiments A1-A68,wherein the first chimeric polypeptide and/or the second chimericpolypeptide further comprises a signal sequence at its N-terminal end.

Embodiment A70

A composition comprising any of the multi-chain chimeric polypeptides ofembodiments A1-A69.

Embodiment A71

The composition of embodiment A70, wherein the composition is apharmaceutical composition.

Embodiment A72

A kit comprising at least one dose of the composition of embodiment A70or A71.

Embodiment A73

A method of stimulating an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments A1-A69 or thecomposition of embodiment A70 or A71.

Embodiment A74

The method of embodiment A73, wherein the immune cell is contacted invitro.

Embodiment A75

The method of embodiment A74, wherein the immune cell was previouslyobtained from a subject.

Embodiment A76

The method of embodiment A75, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment A77

The method of embodiment A73, wherein the immune cell is contacted invivo.

Embodiment A78

The method of any one of embodiments A73-A77, wherein the immune cell isselected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ T cell, aCD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment A79

The method of any one of embodiments A73-A78, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment A80

The method of any one of embodiments A73-A78, wherein the method furthercomprises, after the contacting step, introducing into the immune cell anucleic acid encoding a chimeric antigen-receptor or a recombinantT-cell receptor.

Embodiment A81

The method of any one of embodiments A73-A80, wherein the method furthercomprises administering the immune cell to a subject in need thereof.

Embodiment A82

The method of embodiment A81, wherein the subject has been identified ordiagnosed as having an age-related disease or condition.

Embodiment A83

The method of embodiment A82, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment A84

The method of embodiment A81, wherein the subject has been identified ordiagnosed as having a cancer.

Embodiment A85

The method of embodiment A84, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment A86

The method of embodiment A81, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment A87

The method of embodiment A86, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment A88

A method of inducing or increasing proliferation of an immune cell, themethod comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments A1-A69 or thecomposition of embodiment A70 or A71.

Embodiment A89

The method of embodiment A88, wherein the immune cell is contacted invitro.

Embodiment A90

The method of embodiment A89, wherein the immune cell was previouslyobtained from a subject.

Embodiment A91

The method of embodiment A90, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment A92

The method of embodiment A88, wherein the immune cell is contacted invivo.

Embodiment A93

The method of any one of embodiments A88-A92, wherein the immune cell isselected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ T cell, aCD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment A94

The method of any one of embodiments A88-A93, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment A95

The method of any one of embodiments A88-A93, wherein the method furthercomprises, after the contacting step, introducing into the immune cell anucleic acid encoding a chimeric antigen-receptor or a recombinantT-cell receptor.

Embodiment A96

The method of any one of embodiments A88-A95, wherein the method furthercomprises administering the immune cell to a subject in need thereof.

Embodiment A97

The method of embodiment A96, wherein the subject has been identified ordiagnosed as having an age-related disease or condition.

Embodiment A98

The method of embodiment A97, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment A99

The method of embodiment A96, wherein the subject has been identified ordiagnosed as having a cancer.

Embodiment A100

The method of embodiment A99, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment A101

The method of embodiment A96, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment A102

The method of embodiment A96, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment A103

A method of inducing differentiation of an immune cell into a memory ormemory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments A1-A69 or thecomposition of embodiment A70 or A71.

Embodiment A104

The method of embodiment A103, wherein the immune cell is contacted invitro.

Embodiment A105

The method of embodiment A104, wherein the immune cell was previouslyobtained from a subject.

Embodiment A106

The method of embodiment A105, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment A107

The method of embodiment A103, wherein the immune cell is contacted invivo.

Embodiment A108

The method of any one of embodiments A103-A107, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a Th17 cell, a Th22cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ Tcell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, anatural killer T cell, a mast cell, a macrophage, a neutrophil, adendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment A109

The method of any one of embodiments A103-A108, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment A110

The method of any one of embodiments A103-A108, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment A111

The method of any one of embodiments A103-A110, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment A112

The method of embodiment A111, wherein the subject has been identifiedor diagnosed as having an age-related disease or condition.

Embodiment A113

The method of embodiment A112, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment A114

The method of embodiment A111, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment A115

The method of embodiment A114, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment A116

The method of embodiment A111, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment A117

The method of embodiment A116, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment A118

A method of killing a cancer cell, an infected cell, or a senescent cellin a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of any of the multi-chainchimeric polypeptides of embodiments A1-A69 or the composition ofembodiment A70 or A71.

Embodiment A119

The method of embodiment A118, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment A120

The method of embodiment A119, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment A121

The method of embodiment A118, wherein the subject has been identifiedor diagnosed as having an aging-related disease or condition.

Embodiment A122

The method of embodiment A121, wherein the aging-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment A123

A method of treating a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anyof the multi-chain chimeric polypeptides of embodiments A1-A69 or thecomposition of embodiment A70 or A71.

Embodiment A124

The method of embodiment A123, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment A125

The method of embodiment A124, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment A126

The method of embodiment A123, wherein the subject has been identifiedor diagnosed as having an aging-related disease or condition.

Embodiment A127

The method of embodiment A126, wherein the aging-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment A128

The method of embodiment A123, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment A129

The method of embodiment A128, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment A130

Nucleic acid encoding any of the multi-chain chimeric polypeptides ofany one of embodiments A1-A69.

Embodiment A131

A vector comprising the nucleic acid of embodiment A130.

Embodiment A132

The vector of embodiment A131, wherein the vector is an expressionvector.

Embodiment A133

A cell comprising the nucleic acid of embodiment A130 or the vector ofembodiment A131 or A132.

Embodiment A134

A method of producing a multi-chain chimeric polypeptide, the methodcomprising:

culturing the cell of embodiment A133 in a culture medium underconditions sufficient to result in the production of the multi-chainchimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or theculture medium.

Embodiment A135

A multi-chain chimeric polypeptide produced by the method of embodimentA134.

Embodiment A136

The multi-chain chimeric polypeptide of embodiment A56, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 3.

Embodiment A137

The multi-chain chimeric polypeptide of embodiment A136, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 3.

Embodiment A138

The multi-chain chimeric polypeptide of embodiment A137, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 3.

Embodiment A139

The multi-chain chimeric polypeptide of embodiment A138, wherein thesoluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 3.

Embodiment A140

The multi-chain chimeric polypeptide of embodiment A56, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 4.

Embodiment A141

The multi-chain chimeric polypeptide of embodiment A140, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 4.

Embodiment A142

The multi-chain chimeric polypeptide of embodiment A141, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 4.

Embodiment A143

The multi-chain chimeric polypeptide of embodiment A142, wherein thesoluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 4.

Embodiment A144

The multi-chain chimeric polypeptide of embodiment 56, wherein the humansoluble tissue factor domain does not initiate coagulation.

Embodiment A145

The multi-chain chimeric polypeptide of any one of claims 1-59, whereinthe soluble tissue factor domain comprises or consists of a sequencefrom a wildtype soluble human tissue factor.

B. EXEMPLARY EMBODIMENTS Embodiment B1

A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

-   -   (i) a first target-binding domain;    -   (ii) a soluble tissue factor domain; and    -   (iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

-   -   (i) a second domain of a pair of affinity domains; and    -   (ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptideassociate through the binding of the first domain and the second domainof the pair of affinity domains;

the first target-binding domain and the second targeting-binding domaineach independently bind specifically to a receptor of IL-18 or areceptor of IL-12.

Embodiment B2

The multi-chain chimeric polypeptide of embodiment B1, wherein the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide.

Embodiment B3

The multi-chain chimeric polypeptide of embodiment B 1, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe first target-binding domain and the soluble tissue factor domain inthe first chimeric polypeptide.

Embodiment B4

The multi-chain chimeric polypeptide of any one of embodiments B1-B3,wherein the soluble tissue factor domain and the first domain of thepair of affinity domains directly abut each other in the first chimericpolypeptide.

Embodiment B5

The multi-chain chimeric polypeptide of any one of embodiments B1-B3,wherein the first chimeric polypeptide further comprises a linkersequence between the soluble tissue factor domain and the first domainof the pair of affinity domains in the first chimeric polypeptide.

Embodiment B6

The multi-chain chimeric polypeptide of any one of embodiments B1-B5,wherein the second domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide.

Embodiment B7

The multi-chain chimeric polypeptide of any one of embodiments B1-B5,wherein second chimeric polypeptide further comprises a linker sequencebetween the second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

Embodiment B8

The multi-chain chimeric polypeptide of any one of embodiments B1-B7,wherein the soluble tissue factor domain is a soluble human tissuefactor domain.

Embodiment B9

The multi-chain chimeric polypeptide of embodiment B8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 1.

Embodiment B10

The multi-chain chimeric polypeptide of embodiment B9, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 1.

Embodiment B11

The multi-chain chimeric polypeptide of embodiment B10, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 1.

Embodiment B12

The multi-chain chimeric polypeptide of any one of embodiments B8-B11,wherein the soluble human tissue factor domain does not comprise one ormore of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment B13

The multi-chain chimeric polypeptide of embodiment B12, wherein thesoluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment B14

The multi-chain chimeric polypeptide of any one of embodiments B1-B13,wherein the soluble tissue factor domain is not capable of binding toFactor VIIa.

Embodiment B15

The multi-chain chimeric polypeptide of any one of embodiments B1-B14,wherein the soluble tissue factor domain does not convert inactiveFactor X into Factor Xa.

Embodiment B16

The multi-chain chimeric polypeptide of any one of embodiments B1-B15,wherein the multi-chain chimeric polypeptide does not stimulatecoagulation in a mammal.

Embodiment B17

The multi-chain chimeric polypeptide of any one of embodiments B1-B16,wherein the first chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the first chimericpolypeptide.

Embodiment B18

The multi-chain chimeric polypeptide of any one of embodiments B1-B17,wherein the second chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the second chimericpolypeptide.

Embodiment B19

The multi-chain chimeric polypeptide of any one of embodiments B 1-18,wherein the first chimeric polypeptide and/or the second chimericpolypeptide further comprises a signal sequence at its N-terminal end.

Embodiment B20

The multi-chain chimeric polypeptide of embodiment B19, wherein thesignal sequence comprises SEQ ID NO: 31.

Embodiment B21

The multi-chain chimeric polypeptide of embodiment B20, wherein thesignal sequence is SEQ ID NO: 31.

Embodiment B22

The multi-chain chimeric polypeptide of any one of embodiments B1-B21,wherein the pair of affinity domains is a sushi domain from an alphachain of human IL-15 receptor (IL15Rα) and a soluble IL-15.

Embodiment B23

The multi-chain chimeric polypeptide of embodiment B22, wherein thesoluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment B24

The multi-chain chimeric polypeptide of embodiment B22, wherein thesoluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO:14.

Embodiment B25

The multi-chain chimeric polypeptide of embodiment B24, wherein thesoluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO:14.

Embodiment B26

The multi-chain chimeric polypeptide of embodiment B25, wherein thesoluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO:14.

Embodiment B27

The multi-chain chimeric polypeptide of embodiment B26, wherein thesoluble IL-15 comprises SEQ ID NO: 14.

Embodiment B28

The multi-chain chimeric polypeptide of any one of embodiments B22-B27,wherein the sushi domain of IL15Rα comprises a sushi domain from humanIL15Rα.

Embodiment B29

The multi-chain chimeric polypeptide of embodiment B28, wherein thesushi domain from human IL15Rα comprises a sequence that is 80%identical to SEQ ID NO: 28.

Embodiment B30

The multi-chain chimeric polypeptide of embodiment B29, wherein thesushi domain from human IL15Rα comprises a sequence that is 90%identical to SEQ ID NO: 28.

Embodiment B31

The multi-chain chimeric polypeptide of embodiment B30, wherein thesushi domain from human IL15Rα comprises a sequence that is 95%identical to SEQ ID NO: 28.

Embodiment B32

The multi-chain chimeric polypeptide of embodiment B31, wherein thesushi domain from human IL15Rα comprises SEQ ID NO: 28.

Embodiment B33

The multi-chain chimeric polypeptide of embodiment B28, wherein thesushi domain from human IL15Rα is a mature full-length IL15Rα.

Embodiment B34

The multi-chain chimeric polypeptide of any one of embodiments B1-B21,wherein the pair of affinity domains is selected from the groupconsisting of: barnase and barnstar, a PKA and an AKAP, adapter/dockingtag modules based on mutated RNase I fragments, and SNARE modules basedon interactions of the proteins syntaxin, synaptotagmin, synaptobrevin,and SNAP25.

Embodiment B35

The multi-chain chimeric polypeptide of any one of embodiments B1-B34,wherein one or both of the first target-binding domain and the secondtarget-binding domain is an agonistic antigen-binding domain.

Embodiment B36

The multi-chain chimeric polypeptide of embodiment B35, wherein thefirst target-binding domain and the second target-binding domain areeach agonistic antigen-binding domains.

Embodiment B37

The multi-chain chimeric polypeptide of embodiment B35 or B36, whereinantigen-binding domain comprises a scFv or single-domain antibody.

Embodiment B38

The multi-chain chimeric polypeptide of any one of embodiments B1-B34,wherein one or both of the first target-binding domain and the secondtarget-binding domain is a soluble IL-15 or a soluble IL-18.

Embodiment B39

The multi-chain chimeric polypeptide of embodiment B38, wherein thefirst target-binding domain and the second target-binding domain areeach independently a soluble IL-15 or a soluble IL-18.

Embodiment B40

The multi-chain chimeric polypeptide of any one of embodiments B1-B39,wherein the first target-binding domain and the second target-bindingdomain both bind specifically to a receptor of IL-18 or a receptor ofIL-12.

Embodiment B41

The multi-chain chimeric polypeptide of embodiment B40, wherein thefirst target-binding domain and the second target-binding domain bindspecifically to the same epitope.

Embodiment B42

The multi-chain chimeric polypeptide of embodiment B41, wherein thefirst target-binding domain and the second target-binding domaincomprise the same amino acid sequence.

Embodiment B43

The multi-chain chimeric polypeptide of any one of embodiments B1-B39,wherein the first target-binding domain binds specifically to a receptorfor IL-12, and the second target-binding domain binds specifically to areceptor for IL-18.

Embodiment B44

The multi-chain chimeric polypeptide of any one of embodiments B1-B39,wherein the first target-binding domain binds specifically to a receptorfor IL-18, and the second target-binding domain bind specifically to areceptor for IL-12.

Embodiment B45

The multi-chain chimeric polypeptide of embodiment B44, wherein thefirst target-binding domain comprises a soluble IL-18.

Embodiment B46

The multi-chain chimeric polypeptide of embodiment B45, wherein thesoluble IL-18 is a soluble human IL-18.

Embodiment B47

The multi-chain chimeric polypeptide of embodiment B46, wherein thesoluble human IL-18 comprises a sequence at least 80% identical to SEQID NO: 16.

Embodiment B48

The multi-chain chimeric polypeptide of embodiment B47, wherein thesoluble human IL-18 comprises a sequence at least 90% identical to SEQID NO: 16.

Embodiment B49

The multi-chain chimeric polypeptide of embodiment B48, wherein thesoluble human IL-18 comprises a sequence at least 95% identical to SEQID NO: 16.

Embodiment B50

The multi-chain chimeric polypeptide of embodiment B49, wherein thesoluble human IL-18 comprises a sequence of SEQ ID NO: 16.

Embodiment B51

The multi-chain chimeric polypeptide of any one of embodiments B44-B50,wherein the second target-binding domain comprises a soluble IL-12.

Embodiment B52

The multi-chain chimeric polypeptide of embodiment B51, wherein thesoluble IL-18 is a soluble human IL-12.

Embodiment B53

The multi-chain chimeric polypeptide of embodiment B52, wherein thesoluble human IL-15 comprises a sequence of soluble human IL-12β (p40)and a sequence of soluble human IL-12α (p35).

Embodiment B54

The multi-chain chimeric polypeptide of embodiment B53, wherein thesoluble human IL-15 further comprises a linker sequence between thesequence of soluble IL-12β (p40) and the sequence of soluble humanIL-12α (p35).

Embodiment B55

The multi-chain chimeric polypeptide of embodiment B54, wherein thelinker sequence comprises SEQ ID NO: 7.

Embodiment B56

The multi-chain chimeric polypeptide of any one of embodiments B53-B55,wherein the sequence of soluble human IL-12β (p40) comprises a sequencethat is at least 80% identical to SEQ ID NO: 66.

Embodiment B57

The multi-chain chimeric polypeptide of embodiment B56, wherein thesequence of soluble human IL-12β (p40) comprises a sequence that is atleast 90% identical to SEQ ID NO: 66.

Embodiment B58

The multi-chain chimeric polypeptide of embodiment B57, wherein thesequence of soluble human IL-12β (p40) comprises a sequence that is atleast 95% identical to SEQ ID NO: 66.

Embodiment B59

The multi-chain chimeric polypeptide of embodiment B58, wherein thesequence of soluble human IL-12β (p40) comprises SEQ ID NO: 66.

Embodiment B60

The multi-chain chimeric polypeptide of any one of embodiments B53-B59,wherein the sequence of soluble human IL-12α (p35) comprises a sequencethat is at least 80% identical to SEQ ID NO: 68.

Embodiment B61

The multi-chain chimeric polypeptide of embodiment B60, wherein thesequence of soluble human IL-12α (p35) comprises a sequence that is atleast 90% identical to SEQ ID NO: 68.

Embodiment B62

The mule-chain chimeric polypeptide of embodiment B61, wherein thesequence of soluble human IL-12α (p35) comprises a sequence that is atleast 95% identical to SEQ ID NO: 68.

Embodiment B63

The multi-chain chimeric polypeptide of embodiment B62, wherein thesequence of soluble human IL-12α (p35) comprises SEQ ID NO: 68.

Embodiment B64

The multi-chain chimeric polypeptide of embodiment B1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 70.

Embodiment B65

The multi-chain chimeric polypeptide of embodiment B64, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 70.

Embodiment B66

The multi-chain chimeric polypeptide of embodiment B65, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 70.

Embodiment B67

The multi-chain chimeric polypeptide of embodiment B66, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 70.

Embodiment B68

The multi-chain chimeric polypeptide of embodiment B67, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 72.

Embodiment B69

The multi-chain chimeric polypeptide of any one of embodiments B1 andB64-B68, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 74.

Embodiment B70

The multi-chain chimeric polypeptide of embodiment B69, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 74.

Embodiment B71

The multi-chain chimeric polypeptide of embodiment B70, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 74.

Embodiment B72

The multi-chain chimeric polypeptide of embodiment B71, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 74.

Embodiment B73

The multi-chain chimeric polypeptide of embodiment B72, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 74.

Embodiment B74

The multi-chain chimeric polypeptide of any one of embodiments B1-B63,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domain(s), where at least one of the one ormore additional antigen-binding domain(s) is positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains.

Embodiment B75

The multi-chain chimeric polypeptide of embodiment B74, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe soluble tissue factor domain and the at least one of the one or moreadditional antigen-binding domain(s), and/or a linker sequence betweenthe at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment B76

The multi-chain chimeric polypeptide of any one of embodiments B1-B63,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domains at the N-terminal and/or C-terminalend of the first chimeric polypeptide.

Embodiment B77

The multi-chain chimeric polypeptide of embodiment B76, wherein at leastone of the one or more additional target-binding domains directly abutsthe first domain of the pair of affinity domains in the first chimericpolypeptide.

Embodiment B78

The multi-chain chimeric polypeptide of embodiment B76, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first domain of the pair of affinity domains.

Embodiment B79

The multi-chain chimeric polypeptide of embodiment B76, wherein the atleast one of the one or more additional target-binding domains directlyabuts the first target-binding domain in the first chimeric polypeptide.

Embodiment B80

The multi-chain chimeric polypeptide of embodiment B76, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first target-binding domain.

Embodiment B81

The multi-chain chimeric polypeptide of embodiment B76, wherein at leastone of the one or more additional target-binding domains is disposed atthe N- and/or C-terminus of the first chimeric polypeptide, and at leastone of the one or more additional target-binding domains is positionedbetween the soluble tissue factor domain and the first domain of thepair of affinity domains in the first chimeric polypeptide.

Embodiment B82

The multi-chain chimeric polypeptide of embodiment B81, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the N-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment B83

The multi-chain chimeric polypeptide of embodiment B81, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment B84

The multi-chain chimeric polypeptide of embodiment B81, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the C-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment B85

The multi-chain chimeric polypeptide of embodiment B81, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment B86

The multi-chain chimeric polypeptide of embodiment B81, wherein the atleast one of the one or more additional target-binding domainspositioned between the soluble tissue factor domain and the first domainof the pair of affinity domains, directly abuts the soluble tissuefactor domain and/or the first domain of the pair of affinity domains.

Embodiment B87

The multi-chain chimeric polypeptide of embodiment B81, wherein thefirst chimeric polypeptide further comprises a linker sequence disposed(i) between the soluble tissue factor domain and the at least one of theone or more additional target-binding domains positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains, and/or (ii) between the first domain of the pair ofaffinity domains and the at least one of the one or more additionaltarget-binding domains positioned between the soluble tissue factordomain and the first domain of the pair of affinity domains.

Embodiment B88

The multi-chain chimeric polypeptide of any one of embodiments B1-B63and B74-B87, wherein the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

Embodiment B89

The multi-chain chimeric polypeptide of embodiment B88, wherein at leastone of the one or more additional target-binding domains directly abutsthe second domain of the pair of affinity domains in the second chimericpolypeptide.

Embodiment B90

The multi-chain chimeric polypeptide of embodiment B88, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second domain of the pair of affinity domains in the second chimericpolypeptide.

Embodiment B91

The multi-chain chimeric polypeptide of embodiment B88, wherein at leastone of the one or more additional target-binding domains directly abutsthe second target-binding domain in the second chimeric polypeptide.

Embodiment B92

The multi-chain chimeric polypeptide of embodiment B88, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second target-binding domain in the second chimeric polypeptide.

Embodiment B93

The multi-chain chimeric polypeptide of any one of embodiments B74-B92,wherein two or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains bind specifically to the same antigen.

Embodiment B94

The multi-chain chimeric polypeptide of embodiment B93, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same epitope.

Embodiment B95

The multi-chain chimeric polypeptide of embodiment B94, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains comprisethe same amino acid sequence.

Embodiment B96

The multi-chain chimeric polypeptide of any one of embodiments B74-B92,wherein the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to different antigens.

Embodiment B97

The multi-chain chimeric polypeptide of any one of embodiments B74-B96,wherein the one or more additional antigen-binding domains bindspecifically to a target selected from the group consisting of: CD16a,CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R,IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand ofTGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, aligand of NKG2D, a ligand of NKP30, a ligand for a scMHCI, a ligand fora scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor forIL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, and a receptor for CD28.

Embodiment B98

The multi-chain chimeric polypeptide of any one of embodiments B74-B96,wherein the one or more additional target-binding domains is a solubleinterleukin or cytokine protein.

Embodiment B99

The multi-chain chimeric polypeptide of embodiment B98, wherein thesoluble interleukin, cytokine, or ligand protein is selected from thegroup consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment B100

The multi-chain chimeric polypeptide of any one of embodiments B74-B96,wherein the one or more additional target-binding domains is a solubleinterleukin or cytokine receptor.

Embodiment B101

The multi-chain chimeric polypeptide of embodiment B100, wherein thesoluble receptor is a soluble TGF-β receptor II (TGF-β RII), a solubleTGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a solubleNKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155,a soluble CD122, or a soluble CD28.

Embodiment B102

A composition comprising any of the multi-chain chimeric polypeptides ofembodiments B1-B101.

Embodiment B103

The composition of embodiment B102, wherein the composition is apharmaceutical composition.

Embodiment B104

A kit comprising at least one dose of the composition of embodiment B102or B103.

Embodiment B105

A method of stimulating an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments B1-B101 or thecomposition of embodiment B102 or B103.

Embodiment B106

The method of embodiment B105, wherein the immune cell is contacted invitro.

Embodiment B107

The method of embodiment B106, wherein the immune cell was previouslyobtained from a subject.

Embodiment B108

The method of embodiment B107, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment B109

The method of embodiment B105, wherein the immune cell is contacted invivo.

Embodiment B110

The method of any one of embodiments B105-B109, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ T cell, aCD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment B111

The method of any one of embodiments B105-B110, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment B112

The method of any one of embodiments B105-B110, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment B113

The method of any one of embodiments B105-B112, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment B114

The method of embodiment B113, wherein the subject has been identifiedor diagnosed as having age-related disease or condition.

Embodiment B115

The method of embodiment B114, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment B116

The method of embodiment B113, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment B117

The method of embodiment B116, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment B118

The method of embodiment B113, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment B119

The method of embodiment B118, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment B120

A method of inducing or increasing proliferation of an immune cell, themethod comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments B1-B101 or thecomposition of embodiment B102 or B103.

Embodiment B121

The method of embodiment B120, wherein the immune cell is contacted invitro.

Embodiment B122

The method of embodiment B121, wherein the immune cell was previouslyobtained from a subject.

Embodiment B123

The method of embodiment B122, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment B124

The method of embodiment B120, wherein the immune cell is contacted invivo.

Embodiment B125

The method of any one of embodiments B120-B124, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, aCD4+ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment B126

The method of any one of embodiments B120-B125, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment B127

The method of any one of embodiments B120-B125, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment B128

The method of any one of embodiments B120-B127, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment B129

The method of embodiment B128, wherein the subject has been identifiedor diagnosed as having an age-related disease or condition.

Embodiment B130

The method of embodiment B129, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment B131

The method of embodiment B128, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment B132

The method of embodiment B131, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment B133

The method of embodiment B128, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment B134

The method of embodiment B128, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment B135

A method of inducing differentiation of an immune cell into a memory ormemory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments B1-B101 or thecomposition of embodiment B102 or B103.

Embodiment B136

The method of embodiment B135, wherein the immune cell is contacted invitro.

Embodiment B137

The method of embodiment B136, wherein the immune cell was previouslyobtained from a subject.

Embodiment B138

The method of embodiment B137, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment B139

The method of embodiment B135, wherein the immune cell is contacted invivo.

Embodiment B140

The method of any one of embodiments B135-B139, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a Th17 cell, a Th22cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ Tcell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, anatural killer T cell, a mast cell, a macrophage, a neutrophil, adendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment B141

The method of any one of embodiments B135-B140, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment B142

The method of any one of embodiments B135-B140, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment B143

The method of any one of embodiments B135-B142, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment B144

The method of embodiment B143, wherein the subject has been identifiedor diagnosed as having an age-related disease or condition.

Embodiment B145

The method of embodiment B144, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment B146

The method of embodiment B143, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment B147

The method of embodiment B146, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment B148

The method of embodiment B143, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment B149

The method of embodiment B148, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment B150

A method of killing a cancer cell, an infected cell, or a senescent cellin a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of any of the multi-chainchimeric polypeptides of embodiments B1-B101 or the composition ofembodiment B102 or B103.

Embodiment B151

The method of embodiment B150, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment B152

The method of embodiment B151, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment B153

The method of embodiment B150, wherein the subject has been identifiedor diagnosed as having an aging-related disease or condition.

Embodiment B154

The method of embodiment B153, wherein the aging-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction

Embodiment B155

A method of treating a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anyof the multi-chain chimeric polypeptides of embodiments B1-B101 or thecomposition of embodiment B102 or B103.

Embodiment B156

The method of embodiment B155, wherein the subject has been identifiedor diagnosed as having a cancer or infectious disease.

Embodiment B157

The method of embodiment B156, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment B158

The method of embodiment B155, wherein the subject has been identifiedor diagnosed as having age-related disease or condition.

Embodiment B159

The method of embodiment B158, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment B160

The method of embodiment B155, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment B161

The method of embodiment B160, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment B162

Nucleic acid encoding any of the multi-chain chimeric polypeptides ofany one of embodiments B1-B101.

Embodiment B163

A vector comprising the nucleic acid of embodiment B162.

Embodiment B164

The vector of embodiment B163, wherein the vector is an expressionvector.

Embodiment B165

A cell comprising the nucleic acid of embodiment B162 or the vector ofembodiment B163 or B164.

Embodiment B166

A method of producing a multi-chain chimeric polypeptide, the methodcomprising:

culturing the cell of embodiment B165 in a culture medium underconditions sufficient to result in the production of the multi-chainchimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or theculture medium.

Embodiment B167

A multi-chain chimeric polypeptide produced by the method of embodimentB166.

Embodiment B168

The multi-chain chimeric polypeptide of embodiment B8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 3.

Embodiment B169

The multi-chain chimeric polypeptide of embodiment B168, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 3.

Embodiment B170

The multi-chain chimeric polypeptide of embodiment B169, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 3.

Embodiment B171

The multi-chain chimeric polypeptide of embodiment B170, wherein thesoluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 3.

Embodiment B172

The multi-chain chimeric polypeptide of embodiment B8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 4.

Embodiment B173

The multi-chain chimeric polypeptide of embodiment B172, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 4.

Embodiment B174

The multi-chain chimeric polypeptide of embodiment B173, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 4.

Embodiment B175

The multi-chain chimeric polypeptide of embodiment B174, wherein thesoluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 4.

C. EXEMPLARY EMBODIMENTS Embodiment C1

A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

-   -   (i) a first target-binding domain;    -   (ii) a soluble tissue factor domain; and    -   (iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

-   -   (i) a second domain of a pair of affinity domains; and    -   (ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptideassociate through the binding of the first domain and the second domainof the pair of affinity domains; and

the first target-binding domain and the second targeting-binding domaineach independently bind specifically to a receptor of IL-21 or a ligandof tumor growth factor receptor β II (TGFβRII).

Embodiment C2

The multi-chain chimeric polypeptide of embodiment C1, wherein the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide.

Embodiment C3

The multi-chain chimeric polypeptide of embodiments C1, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe first target-binding domain and the soluble tissue factor domain inthe first chimeric polypeptide.

Embodiment C4

The multi-chain chimeric polypeptide of any one of embodiments C1-C3,wherein the soluble tissue factor domain and the first domain of thepair of affinity domains directly abut each other in the first chimericpolypeptide.

Embodiment C5

The multi-chain chimeric polypeptide of any one of embodiments C1-C3,wherein the first chimeric polypeptide further comprises a linkersequence between the soluble tissue factor domain and the first domainof the pair of affinity domains in the first chimeric polypeptide.

Embodiment C6

The multi-chain chimeric polypeptide of any one of embodiments C1-05,wherein the second domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide.

Embodiment C7

The multi-chain chimeric polypeptide of any one of embodiments C1-05,wherein second chimeric polypeptide further comprises a linker sequencebetween the second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

Embodiment C8

The multi-chain chimeric polypeptide of any one of embodiments C1-C7,wherein the soluble tissue factor domain is a soluble human tissuefactor domain.

Embodiment C9

The multi-chain chimeric polypeptide of embodiment C8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 1.

Embodiment C10

The multi-chain chimeric polypeptide of embodiment C9, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 1.

Embodiment C11

The multi-chain chimeric polypeptide of embodiment C10, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 1.

Embodiment C12

The multi-chain chimeric polypeptide of any one of embodiments C8-C11,wherein the soluble human tissue factor domain does not comprise one ormore of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment C13

The multi-chain chimeric polypeptide of embodiment C12, wherein thesoluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment C14

The multi-chain chimeric polypeptide of any one of embodiments C1-C13,wherein the soluble tissue factor domain is not capable of binding toFactor VIIa.

Embodiment C15

The multi-chain chimeric polypeptide of any one of embodiments C1-C14,wherein the soluble tissue factor domain does not convert inactiveFactor X into Factor Xa.

Embodiment C16

The multi-chain chimeric polypeptide of any one of embodiments C1-C15,wherein the multi-chain chimeric polypeptide does not stimulate bloodcoagulation in a mammal.

Embodiment C17

The multi-chain chimeric polypeptide of any one of embodiments C1-C16,wherein the first chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the first chimericpolypeptide.

Embodiment C18

The multi-chain chimeric polypeptide of any one of embodiments C1-C17,wherein the second chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the second chimericpolypeptide.

Embodiment C19

The multi-chain chimeric polypeptide of any one of embodiments C1-C18,wherein the first chimeric polypeptide and/or the second chimericpolypeptide further comprises a signal sequence at its N-terminal end.

Embodiment C20

The multi-chain chimeric polypeptide of embodiment C19, wherein thesignal sequence comprises SEQ ID NO: 31.

Embodiment C21

The multi-chain chimeric polypeptide of embodiment C20, wherein thesignal sequence is SEQ ID NO: 31.

Embodiment C22

The multi-chain chimeric polypeptide of any one of embodiments C1-C21,wherein the pair of affinity domains is a sushi domain from an alphachain of human IL-15 receptor (IL15Rα and a soluble IL-15.

Embodiment C23

The multi-chain chimeric polypeptide of embodiment C22, wherein thesoluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment C24

The multi-chain chimeric polypeptide of embodiment C22, wherein thesoluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO:14.

Embodiment C25

The multi-chain chimeric polypeptide of embodiment C24, wherein thesoluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO:14.

Embodiment C26

The multi-chain chimeric polypeptide of embodiment C25, wherein thesoluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO:14.

Embodiment C27

The multi-chain chimeric polypeptide of embodiment C26, wherein thesoluble IL-15 comprises SEQ ID NO: 14.

Embodiment C28

The multi-chain chimeric polypeptide of any one of embodiments C22-C27,wherein the sushi domain of IL15Rα comprises a sushi domain from humanIL15Rα.

Embodiment C29

The multi-chain chimeric polypeptide of embodiment C28, wherein thesushi domain from human IL15Rα comprises a sequence that is 80%identical to SEQ ID NO: 28.

Embodiment C30

The multi-chain chimeric polypeptide of embodiment C29, wherein thesushi domain from human IL15Rα comprises a sequence that is 90%identical to SEQ ID NO: 28.

Embodiment C31

The multi-chain chimeric polypeptide of embodiment C30, wherein thesushi domain from human IL15Rα comprises a sequence that is 95%identical to SEQ ID NO: 28.

Embodiment C32

The multi-chain chimeric polypeptide of embodiment C31, wherein thesushi domain from human IL15Rα comprises SEQ ID NO: 28.

Embodiment C33

The multi-chain chimeric polypeptide of embodiment C28, wherein thesushi domain from human IL15Rα is a mature full-length IL15Rα.

Embodiment C34

The multi-chain chimeric polypeptide of any one of embodiments C1-C21,wherein the pair of affinity domains is selected from the groupconsisting of: barnase and barnstar, a PKA and an AKAP, adapter/dockingtag modules based on mutated RNase I fragments, and SNARE modules basedon interactions of the proteins syntaxin, synaptotagmin, synaptobrevin,and SNAP25.

Embodiment C35

The multi-chain chimeric polypeptide of any one of embodiments C1-C34,wherein one or both of the first target-binding domain and the secondtarget-binding domain is an antigen-binding domain.

Embodiment C36

The multi-chain chimeric polypeptide of embodiment C35, wherein thefirst target-binding domain and the second target-binding domain areantigen-binding domains.

Embodiment C37

The multi-chain chimeric polypeptide of embodiment C35 or C36, whereinantigen-binding domain comprises a scFv or single-domain antibody.

Embodiment C38

The multi-chain chimeric polypeptide of any one of embodiments C1-C34,wherein one or both of the first target-binding domain and the secondtarget-binding domain is a soluble IL-21 or a soluble TGFβRII.

Embodiment C39

The multi-chain chimeric polypeptide of any one of embodiments C1-C38,wherein the first target-binding domain and the second target-bindingdomain both bind specifically to a receptor of IL-21 or a ligand ofTGFβRII.

Embodiment C40

The multi-chain chimeric polypeptide of embodiment C39, wherein thefirst target-binding domain and the second target-binding domain bindspecifically to the same epitope.

Embodiment C41

The multi-chain chimeric polypeptide of embodiment C40, wherein thefirst target-binding domain and the second target-binding domaincomprise the same amino acid sequence.

Embodiment C42

The multi-chain chimeric polypeptide of any one of embodiments C1-C38,wherein the first target-binding domain binds specifically to a ligandof TGFβRII, and the second target-binding domain binds specifically to areceptor for IL-21.

Embodiment C43

The multi-chain chimeric polypeptide of any one of embodiments C1-C38,wherein the first target-binding domain binds specifically to a receptorfor IL-21, and the second target-binding domain bind specifically to aligand of TGFβRII.

Embodiment C44

The multi-chain chimeric polypeptide of embodiment C43, wherein thefirst target-binding domain comprises a soluble IL-21.

Embodiment C45

The multi-chain chimeric polypeptide of embodiment C44, wherein thesoluble IL-21 is a soluble human IL-21.

Embodiment C46

The multi-chain chimeric polypeptide of embodiment C45, wherein thesoluble human IL-21 comprises a sequence at least 80% identical to SEQID NO: 78.

Embodiment C47

The multi-chain chimeric polypeptide of embodiment C46, wherein thesoluble human IL-21 comprises a sequence at least 90% identical to SEQID NO: 78.

Embodiment C48

The multi-chain chimeric polypeptide of embodiment C47, wherein thesoluble human IL-21 comprises a sequence at least 95% identical to SEQID NO: 78.

Embodiment C49

The multi-chain chimeric polypeptide of embodiment C48, wherein thesoluble human IL-21 comprises a sequence of SEQ ID NO: 78.

Embodiment C50

The multi-chain chimeric polypeptide of any one of embodiments C43-C49,wherein the second target-binding domain comprises a soluble TGFβRII.

Embodiment C51

The multi-chain chimeric polypeptide of embodiment C50, wherein thesoluble TGFβRII is a soluble human TGFβRII.

Embodiment C52

The multi-chain chimeric polypeptide of embodiment C51, wherein thesoluble human TGFβRII comprises a first sequence of soluble humanTGFβRII and a second sequence of soluble human TGFβRII.

Embodiment C53

The multi-chain chimeric polypeptide of embodiment C52, wherein thesoluble human TGFβRII further comprises a linker sequence between thefirst sequence of soluble human TGFβRII and the second sequence ofsoluble human TGFβRII.

Embodiment C54

The multi-chain chimeric polypeptide of embodiment C53, wherein thelinker sequence comprises SEQ ID NO: 7.

Embodiment C55

The multi-chain chimeric polypeptide of any one of embodiments C52-054,wherein the first sequence of soluble human TGFβRII comprises a sequencethat is at least 80% identical to SEQ ID NO: 80.

Embodiment C56

The multi-chain chimeric polypeptide of embodiment C55, wherein thefirst sequence of soluble human TGFβRII comprises a sequence that is atleast 90% identical to SEQ ID NO: 80.

Embodiment C57

The multi-chain chimeric polypeptide of embodiment C56, wherein thefirst sequence of soluble human TGFβRII comprises a sequence that is atleast 95% identical to SEQ ID NO: 80.

Embodiment C58

The multi-chain chimeric polypeptide of embodiment C57, wherein thefirst sequence of soluble human TGFβRII comprises SEQ ID NO: 80.

Embodiment C59

The multi-chain chimeric polypeptide of any one of embodiments C52-058,wherein the second sequence of soluble human TGFβRII comprises asequence that is at least 80% identical to SEQ ID NO: 81.

Embodiment C60

The multi-chain chimeric polypeptide of embodiment C59, wherein thesecond sequence of soluble human TGFβRII comprises a sequence that is atleast 90% identical to SEQ ID NO: 81.

Embodiment C61

The mule-chain chimeric polypeptide of embodiment C60, wherein thesecond sequence of soluble human TGFβRII comprises a sequence that is atleast 95% identical to SEQ ID NO: 81.

Embodiment C62

The multi-chain chimeric polypeptide of embodiment C61, wherein thesecond sequence of soluble human TGFβRII comprises SEQ ID NO: 81.

Embodiment C63

The multi-chain chimeric polypeptide of embodiment C1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 86.

Embodiment C64

The multi-chain chimeric polypeptide of embodiment C63, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 86.

Embodiment C65

The multi-chain chimeric polypeptide of embodiment C64, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 86.

Embodiment C66

The multi-chain chimeric polypeptide of embodiment C65, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 86.

Embodiment C67

The multi-chain chimeric polypeptide of embodiment C66, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 88.

Embodiment C68

The multi-chain chimeric polypeptide of any one of embodiments C1 andC63-C67, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 90.

Embodiment C69

The multi-chain chimeric polypeptide of embodiment C68, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 90.

Embodiment C70

The multi-chain chimeric polypeptide of embodiment C69, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 90.

Embodiment C71

The multi-chain chimeric polypeptide of embodiment C70, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 90.

Embodiment C72

The multi-chain chimeric polypeptide of embodiment C71, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 92.

Embodiment C73

The multi-chain chimeric polypeptide of any one of embodiments C1-C62,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domain(s), where at least one of the one ormore additional antigen-binding domain(s) is positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains.

Embodiment C74

The multi-chain chimeric polypeptide of embodiment C73, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe soluble tissue factor domain and the at least one of the one or moreadditional antigen-binding domain(s), and/or a linker sequence betweenthe at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment C75

The multi-chain chimeric polypeptide of any one of embodiments C1-C62,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domains at the N-terminal and/or C-terminalend of the first chimeric polypeptide.

Embodiment C76

The multi-chain chimeric polypeptide of embodiment C75, wherein at leastone of the one or more additional target-binding domains directly abutsthe first domain of the pair of affinity domains in the first chimericpolypeptide.

Embodiment C77

The multi-chain chimeric polypeptide of embodiment C75, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first domain of the pair of affinity domains.

Embodiment C78

The multi-chain chimeric polypeptide of embodiment C75, wherein the atleast one of the one or more additional target-binding domains directlyabuts the first target-binding domain in the first chimeric polypeptide.

Embodiment C79

The multi-chain chimeric polypeptide of embodiment C75, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first target-binding domain.

Embodiment C80

The multi-chain chimeric polypeptide of embodiment C75, wherein at leastone of the one or more additional target-binding domains is disposed atthe N- and/or C-terminus of the first chimeric polypeptide, and at leastone of the one or more additional target-binding domains is positionedbetween the soluble tissue factor domain and the first domain of thepair of affinity domains in the first chimeric polypeptide.

Embodiment C81

The multi-chain chimeric polypeptide of embodiment C80, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the N-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment C82

The multi-chain chimeric polypeptide of embodiment C80, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment C83

The multi-chain chimeric polypeptide of embodiment C80, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the C-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment C84

The multi-chain chimeric polypeptide of embodiment C80, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment C85

The multi-chain chimeric polypeptide of embodiment C80, wherein the atleast one of the one or more additional target-binding domainspositioned between the soluble tissue factor domain and the first domainof the pair of affinity domains, directly abuts the soluble tissuefactor domain and/or the first domain of the pair of affinity domains.

Embodiment C86

The multi-chain chimeric polypeptide of embodiment C80, wherein thefirst chimeric polypeptide further comprises a linker sequence disposed(i) between the soluble tissue factor domain and the at least one of theone or more additional target-binding domains positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains, and/or (ii) between the first domain of the pair ofaffinity domains and the at least one of the one or more additionaltarget-binding domains positioned between the soluble tissue factordomain and the first domain of the pair of affinity domains.

Embodiment C87

The multi-chain chimeric polypeptide of any one of embodiments C1-C62and C73-C86, wherein the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

Embodiment C88

The multi-chain chimeric polypeptide of embodiment C87, wherein at leastone of the one or more additional target-binding domains directly abutsthe second domain of the pair of affinity domains in the second chimericpolypeptide.

Embodiment C89

The multi-chain chimeric polypeptide of embodiment C87, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second domain of the pair of affinity domains in the second chimericpolypeptide.

Embodiment C90

The multi-chain chimeric polypeptide of embodiment C87, wherein at leastone of the one or more additional target-binding domains directly abutsthe second target-binding domain in the second chimeric polypeptide.

Embodiment C91

The multi-chain chimeric polypeptide of embodiment C87, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second target-binding domain in the second chimeric polypeptide.

Embodiment C92

The multi-chain chimeric polypeptide of any one of embodiments C73-C91,wherein two or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains bind specifically to the same antigen.

Embodiment C93

The multi-chain chimeric polypeptide of embodiment C92, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same epitope.

Embodiment C94

The multi-chain chimeric polypeptide of embodiment C93, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains comprisethe same amino acid sequence.

Embodiment C95

The multi-chain chimeric polypeptide of any one of embodiments C73-C91,wherein the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to different antigens.

Embodiment C96

The multi-chain chimeric polypeptide of any one of embodiments C73-C95,wherein the one or more additional antigen-binding domains bindspecifically to a target selected from the group consisting of: CD16a,CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R,IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,CD155, PDGF-D, a ligand of TGF-β receptor II (TGF-RII), a ligand ofTGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, aligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand fora scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor forIL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, and a receptor for CD28.

Embodiment C97

The multi-chain chimeric polypeptide of any one of embodiments C73-C95,wherein the one or more additional target-binding domains is a solubleinterleukin or cytokine protein.

Embodiment C98

The multi-chain chimeric polypeptide of embodiment C97, wherein thesoluble interleukin, cytokine, or ligand protein is selected from thegroup consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment C99

The multi-chain chimeric polypeptide of any one of embodiments C73-C95,wherein the one or more additional target-binding domains is a solubleinterleukin or cytokine receptor.

Embodiment C100

The multi-chain chimeric polypeptide of embodiment C99, wherein thesoluble receptor is a soluble TGF-β receptor II (TGF-β RII), a solubleTGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a solubleNKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155,or a soluble CD28.

Embodiment C101

A composition comprising any of the multi-chain chimeric polypeptides ofembodiments C1-C100.

Embodiment C102

The composition of embodiment C101, wherein the composition is apharmaceutical composition.

Embodiment C103

A kit comprising at least one dose of the composition of embodiment C101or C102.

Embodiment C104

A method of stimulating an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments C1-C100 or thecomposition of embodiment C101 or C102.

Embodiment C105

The method of embodiment C104, wherein the immune cell is contacted invitro.

Embodiment C106

The method of embodiment C105, wherein the immune cell was previouslyobtained from a subject.

Embodiment C107

The method of embodiment C106, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment C108

The method of embodiment C104, wherein the immune cell is contacted invivo.

Embodiment C109

The method of any one of embodiments C104-C108, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ T cell, aCD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment C110

The method of any one of embodiments C104-C109, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment C111

The method of any one of embodiments C104-C109, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment C112

The method of any one of embodiments C104-C111, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment C113

The method of embodiment C112, wherein the subject has been identifiedor diagnosed as having age-related disease or condition.

Embodiment C114

The method of embodiment C113, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment C115

The method of embodiment C112, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment C116

The method of embodiment C115, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment C117

The method of embodiment C112, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment C118

The method of embodiment C117, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment C119

A method of inducing or increasing proliferation of an immune cell, themethod comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments C1-C100 or thecomposition of embodiment C101 or C102.

Embodiment C120

The method of embodiment C119, wherein the immune cell is contacted invitro.

Embodiment C121

The method of embodiment C120, wherein the immune cell was previouslyobtained from a subject.

Embodiment C122

The method of embodiment C121, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment C123

The method of embodiment C119, wherein the immune cell is contacted invivo.

Embodiment C124

The method of any one of embodiments C119-C123, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, aCD4+ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment C125

The method of any one of embodiments C119-C124, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment C126

The method of any one of embodiments C119-C124, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment C127

The method of any one of embodiments C119-C126, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment C128

The method of embodiment C127, wherein the subject has been identifiedor diagnosed as having an age-related disease or condition.

Embodiment C129

The method of embodiment C128, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment C130

The method of embodiment C127, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment C131

The method of embodiment C130, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment C132

The method of embodiment C127, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment C133

The method of embodiment C127, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment C134

A method of inducing differentiation of an immune cell into a memory ormemory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments C1-C100 or thecomposition of embodiment C101 or C102.

Embodiment C135

The method of embodiment C134, wherein the immune cell is contacted invitro.

Embodiment C136

The method of embodiment C135, wherein the immune cell was previouslyobtained from a subject.

Embodiment C137

The method of embodiment C136, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment C138

The method of embodiment C134, wherein the immune cell is contacted invivo.

Embodiment C139

The method of any one of embodiments C134-C138, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a Th17 cell, a Th22cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ Tcell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, anatural killer T cell, a mast cell, a macrophage, a neutrophil, adendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment C140

The method of any one of embodiments C134-C139, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment C141

The method of any one of embodiments C134-C139, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment C142

The method of any one of embodiments C134-C141, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment C143

The method of embodiment C142, wherein the subject has been identifiedor diagnosed as having an age-related disease or condition.

Embodiment C144

The method of embodiment C143, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment C145

The method of embodiment C142, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment C146

The method of embodiment C145, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment C147

The method of embodiment C142, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment C148

The method of embodiment C147, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment C149

A method of killing a cancer cell, an infected cell, or a senescent cellin a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of any of the multi-chainchimeric polypeptides of embodiments C1-C100 or the composition ofembodiment C101 or C102.

Embodiment C150

The method of embodiment C149, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment C151

The method of embodiment C150, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment C152

The method of embodiment C149, wherein the subject has been identifiedor diagnosed as having an aging-related disease or condition.

Embodiment C153

The method of embodiment C152, wherein the aging-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction

Embodiment C154

A method of treating a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anyof the multi-chain chimeric polypeptides of embodiments C1-C100 or thecomposition of embodiment C101 or C102.

Embodiment C155

The method of embodiment C154, wherein the subject has been identifiedor diagnosed as having a cancer or infectious disease.

Embodiment C156

The method of embodiment C157, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment C157

The method of embodiment C154, wherein the subject has been identifiedor diagnosed as having age-related disease or condition.

Embodiment C158

The method of embodiment C157, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment C159

The method of embodiment C154, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment C160

The method of embodiment C159, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment C161

Nucleic acid encoding any of the multi-chain chimeric polypeptides ofany one of embodiments C1-C100.

Embodiment C162

A vector comprising the nucleic acid of embodiment C161.

Embodiment C163

The vector of embodiment C162, wherein the vector is an expressionvector.

Embodiment C164

A cell comprising the nucleic acid of embodiment C161 or the vector ofembodiment C162 or C163.

Embodiment C165

A method of producing a multi-chain chimeric polypeptide, the methodcomprising:

culturing the cell of embodiment C164 in a culture medium underconditions sufficient to result in the production of the multi-chainchimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or theculture medium.

Embodiment C166

A multi-chain chimeric polypeptide produced by the method of embodimentC165.

Embodiment C167

The multi-chain chimeric polypeptide of embodiment C12, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 3.

Embodiment C168

The multi-chain chimeric polypeptide of embodiment C167, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 3.

Embodiment C169

The multi-chain chimeric polypeptide of embodiment C168, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 3.

Embodiment C170

The multi-chain chimeric polypeptide of embodiment C169, wherein thesoluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 3.

Embodiment C171

The multi-chain chimeric polypeptide of embodiment C12, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 4.

Embodiment C172

The multi-chain chimeric polypeptide of embodiment C171, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 4.

Embodiment C173

The multi-chain chimeric polypeptide of embodiment C172, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 4.

Embodiment C174

The multi-chain chimeric polypeptide of embodiment C173, wherein thesoluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 4.

D. EXEMPLARY EMBODIMENTS Embodiment D1

A multi-chain chimeric polypeptide comprising:

(c) a first chimeric polypeptide comprising:

-   -   (i) a first target-binding domain;    -   (ii) a soluble tissue factor domain; and    -   (iii) a first domain of a pair of affinity domains;

(d) a second chimeric polypeptide comprising:

-   -   (i) a second domain of a pair of affinity domains; and    -   (ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptideassociate through the binding of the first domain and the second domainof the pair of affinity domains;

the first target-binding domain and the second targeting-binding domaineach independently bind specifically to a receptor of IL-21 or areceptor of IL-7.

Embodiment D2

The multi-chain chimeric polypeptide of embodiment D1, wherein the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide.

Embodiment D3

The multi-chain chimeric polypeptide of embodiment D1, wherein the firstchimeric polypeptide further comprises a linker sequence between thefirst target-binding domain and the soluble tissue factor domain in thefirst chimeric polypeptide.

Embodiment D4

The multi-chain chimeric polypeptide of any one of embodiments D1-D3,wherein the soluble tissue factor domain and the first domain of thepair of affinity domains directly abut each other in the first chimericpolypeptide.

Embodiment D5

The multi-chain chimeric polypeptide of any one of embodiments D1-D3,wherein the first chimeric polypeptide further comprises a linkersequence between the soluble tissue factor domain and the first domainof the pair of affinity domains in the first chimeric polypeptide.

Embodiment D6

The multi-chain chimeric polypeptide of any one of embodiments D1-D5,wherein the second domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide.

Embodiment D7

The multi-chain chimeric polypeptide of any one of embodiments D1-D5,wherein second chimeric polypeptide further comprises a linker sequencebetween the second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

Embodiment D8

The multi-chain chimeric polypeptide of any one of embodiments D1-D7,wherein the soluble tissue factor domain is a soluble human tissuefactor domain.

Embodiment D9

The multi-chain chimeric polypeptide of embodiment D8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 1.

Embodiment D10

The multi-chain chimeric polypeptide of embodiment D9, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 1.

Embodiment D11

The multi-chain chimeric polypeptide of embodiment D10, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 1.

Embodiment D12

The multi-chain chimeric polypeptide of embodiment D11, wherein thesoluble human tissue factor domain comprises SEQ ID NO: 1.

Embodiment D13

The multi-chain chimeric polypeptide of embodiment D8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 3.

Embodiment D14

The multi-chain chimeric polypeptide of embodiment D13, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 3.

Embodiment D15

The multi-chain chimeric polypeptide of embodiment D14, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 3.

Embodiment D16

The multi-chain chimeric polypeptide of embodiment D15, wherein thesoluble human tissue factor domain comprises SEQ ID NO: 3.

Embodiment D17

The multi-chain chimeric polypeptide of embodiment D8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 4.

Embodiment D18

The multi-chain chimeric polypeptide of embodiment D17, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 4.

Embodiment D19

The multi-chain chimeric polypeptide of embodiment D18, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 4.

Embodiment D20

The multi-chain chimeric polypeptide of embodiment D19, wherein thesoluble human tissue factor domain comprises SEQ ID NO: 4.

Embodiment D21

The multi-chain chimeric polypeptide of any one of embodiments D8-D11,D13-D15, and D17-D19, wherein the soluble human tissue factor domaindoes not comprise one or more of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment D22

The multi-chain chimeric polypeptide of embodiment D21, wherein thesoluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment D23

The multi-chain chimeric polypeptide of any one of embodiments D1-D22,wherein the soluble tissue factor domain is not capable of binding toFactor VIIa.

Embodiment D24

The multi-chain chimeric polypeptide of any one of embodiments D1-D23,wherein the soluble tissue factor domain does not convert inactiveFactor X into Factor Xa.

Embodiment D25

The multi-chain chimeric polypeptide of any one of embodiments D1-D24,wherein the multi-chain chimeric polypeptide does not stimulatecoagulation in a mammal.

Embodiment D26

The multi-chain chimeric polypeptide of any one of embodiments D1-D25,wherein the first chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the first chimericpolypeptide.

Embodiment D27

The multi-chain chimeric polypeptide of any one of embodiments D1-D26,wherein the second chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the second chimericpolypeptide.

Embodiment D28

The multi-chain chimeric polypeptide of any one of embodiments D1-D27,wherein the first chimeric polypeptide and/or the second chimericpolypeptide further comprises a signal sequence at its N-terminal end.

Embodiment D29

The multi-chain chimeric polypeptide of embodiment D28, wherein thesignal sequence comprises SEQ ID NO: 31.

Embodiment D30

The multi-chain chimeric polypeptide of embodiment D28, wherein thesignal sequence is SEQ ID NO: 223.

Embodiment D31

The multi-chain chimeric polypeptide of any one of embodiments D1-D30,wherein the pair of affinity domains is a sushi domain from an alphachain of human IL-15 receptor (IL15Rα) and a soluble IL-15.

Embodiment D32

The multi-chain chimeric polypeptide of embodiment D31, wherein thesoluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment D33

The multi-chain chimeric polypeptide of embodiment D31, wherein thesoluble IL-15 comprises a sequence that is at least 80% identical to SEQID NO: 14.

Embodiment D34

The multi-chain chimeric polypeptide of embodiment D33, wherein thesoluble IL-15 comprises a sequence that is at least 90% identical to SEQID NO: 14.

Embodiment D35

The multi-chain chimeric polypeptide of embodiment D34, wherein thesoluble IL-15 comprises a sequence that is at least 95% identical to SEQID NO: 14.

Embodiment D36

The multi-chain chimeric polypeptide of embodiment D35, wherein thesoluble IL-15 comprises SEQ ID NO: 14.

Embodiment D37

The multi-chain chimeric polypeptide of any one of embodiments D31-D36,wherein the sushi domain of IL15Rα comprises a sushi domain from humanIL15Rα.

Embodiment D38

The multi-chain chimeric polypeptide of embodiment D37, wherein thesushi domain from human IL15Rα comprises a sequence that is at least 80%identical to SEQ ID NO: 28.

Embodiment D39

The multi-chain chimeric polypeptide of embodiment D38, wherein thesushi domain from human IL15Rα comprises a sequence that is at least 90%identical to SEQ ID NO: 28.

Embodiment D40

The multi-chain chimeric polypeptide of embodiment D39, wherein thesushi domain from human IL15Rα comprises a sequence that is at least 95%identical to SEQ ID NO: 28.

Embodiment D41

The multi-chain chimeric polypeptide of embodiment D40, wherein thesushi domain from human IL15Rα comprises SEQ ID NO: 28.

Embodiment D42

The multi-chain chimeric polypeptide of embodiment D37, wherein thesushi domain from human IL15Rα is a mature full-length IL15Rα.

Embodiment D43

The multi-chain chimeric polypeptide of any one of embodiments D1-D30,wherein the pair of affinity domains is selected from the groupconsisting of: barnase and barnstar, a PKA and an AKAP, adapter/dockingtag modules based on mutated RNase I fragments, and SNARE modules basedon interactions of the proteins syntaxin, synaptotagmin, synaptobrevin,and SNAP25.

Embodiment D44

The multi-chain chimeric polypeptide of any one of embodiments D1-D43,wherein one or both of the first target-binding domain and the secondtarget-binding domain is an agonistic antigen-binding domain.

Embodiment D45

The multi-chain chimeric polypeptide of embodiment D44, wherein thefirst target-binding domain and the second target-binding domain areeach agonistic antigen-binding domains.

Embodiment D46

The multi-chain chimeric polypeptide of embodiment D44 or D45, whereinantigen-binding domain comprises a scFv or single-domain antibody.

Embodiment D47

The multi-chain chimeric polypeptide of any one of embodiments D1-D43,wherein one or both of the first target-binding domain and the secondtarget-binding domain is a soluble IL-21 or a soluble IL-7.

Embodiment D48

The multi-chain chimeric polypeptide of embodiment D47, wherein thefirst target-binding domain and the second target-binding domain areeach independently a soluble IL-21 or a soluble IL-7.

Embodiment D49

The multi-chain chimeric polypeptide of any one of embodiments D1-D48,wherein the first target-binding domain and the second target-bindingdomain both bind specifically to a receptor of IL-21 or a receptor ofIL-7.

Embodiment D50

The multi-chain chimeric polypeptide of embodiment D49, wherein thefirst target-binding domain and the second target-binding domain bindspecifically to the same epitope.

Embodiment D51

The multi-chain chimeric polypeptide of embodiment D50, wherein thefirst target-binding domain and the second target-binding domaincomprise the same amino acid sequence.

Embodiment D52

The multi-chain chimeric polypeptide of any one of embodiments D1-D48,wherein the first target-binding domain binds specifically to a receptorfor IL-21, and the second target-binding domain binds specifically to areceptor for IL-7.

Embodiment D53

The multi-chain chimeric polypeptide of any one of embodiments D1-D48,wherein the first target-binding domain binds specifically to a receptorfor IL-7, and the second target-binding domain bind specifically to areceptor for IL-21.

Embodiment D54

The multi-chain chimeric polypeptide of embodiment D53, wherein thefirst target-binding domain comprises a soluble IL-21.

Embodiment D55

The multi-chain chimeric polypeptide of embodiment D54, wherein thesoluble IL-21 is a soluble human IL-21.

Embodiment D56

The multi-chain chimeric polypeptide of embodiment D55, wherein thesoluble human IL-21 comprises a sequence at least 80% identical to SEQID NO: 78.

Embodiment D57

The multi-chain chimeric polypeptide of embodiment D56, wherein thesoluble human IL-21 comprises a sequence at least 90% identical to SEQID NO: 78.

Embodiment D58

The multi-chain chimeric polypeptide of embodiment D57, wherein thesoluble human IL-21 comprises a sequence at least 95% identical to SEQID NO: 78.

Embodiment D59

The multi-chain chimeric polypeptide of embodiment D58, wherein thesoluble human IL-21 comprises a sequence of SEQ ID NO: 78.

Embodiment D60

The multi-chain chimeric polypeptide of any one of embodiments D53-D59,wherein the second target-binding domain comprises a soluble IL-7.

Embodiment D61

The multi-chain chimeric polypeptide of embodiment D60, wherein thesoluble IL-7 is a soluble human IL-7.

Embodiment D62

The multi-chain chimeric polypeptide of embodiment D61, wherein thesoluble human IL-7 comprises a sequence at least 80% identical to SEQ IDNO: 11.

Embodiment D63

The multi-chain chimeric polypeptide of embodiment D62, wherein thesoluble human IL-7 comprises a sequence at least 90% identical to SEQ IDNO: 11.

Embodiment D64

The multi-chain chimeric polypeptide of embodiment D63, wherein thesoluble human IL-7 comprises a sequence at least 95% identical to SEQ IDNO: 11.

Embodiment D65

The multi-chain chimeric polypeptide of embodiment D64, wherein thesoluble human IL-7 comprises a sequence of SEQ ID NO: 11.

Embodiment D66

The multi-chain chimeric polypeptide of embodiment D1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 104.

Embodiment D67

The multi-chain chimeric polypeptide of embodiment D66, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 104.

Embodiment D68

The multi-chain chimeric polypeptide of embodiment D67, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 104.

Embodiment D69

The multi-chain chimeric polypeptide of embodiment D68, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 104.

Embodiment D70

The multi-chain chimeric polypeptide of embodiment D69, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 106.

Embodiment D71

The multi-chain chimeric polypeptide of any one of embodiments D1 andD66-D70, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 108.

Embodiment D72

The multi-chain chimeric polypeptide of embodiment D71, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 108.

Embodiment D73

The multi-chain chimeric polypeptide of embodiment D72, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 108.

Embodiment D74

The multi-chain chimeric polypeptide of embodiment D73, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 108.

Embodiment D75

The multi-chain chimeric polypeptide of embodiment D74, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 110.

Embodiment D76

The multi-chain chimeric polypeptide of embodiment D1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 96.

Embodiment D77

The multi-chain chimeric polypeptide of embodiment D76, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 96.

Embodiment D78

The multi-chain chimeric polypeptide of embodiment D77, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 96.

Embodiment D79

The multi-chain chimeric polypeptide of embodiment D68, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 96.

Embodiment D80

The multi-chain chimeric polypeptide of embodiment D69, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 98.

Embodiment D81

The multi-chain chimeric polypeptide of any one of embodiments D1 andD76-D80, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 100.

Embodiment D82

The multi-chain chimeric polypeptide of embodiment D81, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 100.

Embodiment D83

The multi-chain chimeric polypeptide of embodiment D82, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 100.

Embodiment D84

The multi-chain chimeric polypeptide of embodiment D83, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 100.

Embodiment D85

The multi-chain chimeric polypeptide of embodiment D84, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 106.

Embodiment D86

The multi-chain chimeric polypeptide of any one of embodiments D1-D65,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domain(s), where at least one of the one ormore additional antigen-binding domain(s) is positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains.

Embodiment D87

The multi-chain chimeric polypeptide of embodiment D86, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe soluble tissue factor domain and the at least one of the one or moreadditional antigen-binding domain(s), and/or a linker sequence betweenthe at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment D88

The multi-chain chimeric polypeptide of any one of embodiments D1-D65,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domains at the N-terminal and/or C-terminalend of the first chimeric polypeptide.

Embodiment D89

The multi-chain chimeric polypeptide of embodiment D88, wherein at leastone of the one or more additional target-binding domains directly abutsthe first domain of the pair of affinity domains in the first chimericpolypeptide.

Embodiment D90

The multi-chain chimeric polypeptide of embodiment D88, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first domain of the pair of affinity domains.

Embodiment D91

The multi-chain chimeric polypeptide of embodiment D88, wherein the atleast one of the one or more additional target-binding domains directlyabuts the first target-binding domain in the first chimeric polypeptide.

Embodiment D92

The multi-chain chimeric polypeptide of embodiment D88, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first target-binding domain.

Embodiment D93

The multi-chain chimeric polypeptide of embodiment D88, wherein at leastone of the one or more additional target-binding domains is disposed atthe N- and/or C-terminus of the first chimeric polypeptide, and at leastone of the one or more additional target-binding domains is positionedbetween the soluble tissue factor domain and the first domain of thepair of affinity domains in the first chimeric polypeptide.

Embodiment D94

The multi-chain chimeric polypeptide of embodiment D93, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the N-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment D95

The multi-chain chimeric polypeptide of embodiment D93, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment D96

The multi-chain chimeric polypeptide of embodiment D93, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the C-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment D97

The multi-chain chimeric polypeptide of embodiment D93, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment D98

The multi-chain chimeric polypeptide of embodiment D93, wherein the atleast one of the one or more additional target-binding domainspositioned between the soluble tissue factor domain and the first domainof the pair of affinity domains, directly abuts the soluble tissuefactor domain and/or the first domain of the pair of affinity domains.

Embodiment D99

The multi-chain chimeric polypeptide of embodiment D93, wherein thefirst chimeric polypeptide further comprises a linker sequence disposed(i) between the soluble tissue factor domain and the at least one of theone or more additional target-binding domains positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains, and/or (ii) between the first domain of the pair ofaffinity domains and the at least one of the one or more additionaltarget-binding domains positioned between the soluble tissue factordomain and the first domain of the pair of affinity domains.

Embodiment D100

The multi-chain chimeric polypeptide of any one of embodiments D1-D65and D86-D99, wherein the second chimeric polypeptide further comprisesone or more additional target-binding domains at the N-terminal end orthe C-terminal end of the second chimeric polypeptide.

Embodiment D101

The multi-chain chimeric polypeptide of embodiment D100, wherein atleast one of the one or more additional target-binding domains directlyabuts the second domain of the pair of affinity domains in the secondchimeric polypeptide.

Embodiment D102

The multi-chain chimeric polypeptide of embodiment D100, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second domain of the pair of affinity domains in the second chimericpolypeptide.

Embodiment D103

The multi-chain chimeric polypeptide of embodiment D100, wherein atleast one of the one or more additional target-binding domains directlyabuts the second target-binding domain in the second chimericpolypeptide.

Embodiment D104

The multi-chain chimeric polypeptide of embodiment D100, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second target-binding domain in the second chimeric polypeptide.

Embodiment D105

The multi-chain chimeric polypeptide of any one of embodiments D86-D104,wherein two or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains bind specifically to the same antigen.

Embodiment D106

The multi-chain chimeric polypeptide of embodiment D105, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same epitope.

Embodiment D107

The multi-chain chimeric polypeptide of embodiment D106, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains comprisethe same amino acid sequence.

Embodiment D108

The multi-chain chimeric polypeptide of any one of embodiments D86-D104,wherein the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to different antigens.

Embodiment D109

The multi-chain chimeric polypeptide of any one of embodiments D86-D108,wherein the one or more additional antigen-binding domains bindspecifically to a target selected from the group consisting of: CD16a,CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R,IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2,CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand ofTGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, aligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand fora scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor forIL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, and a receptor for CD28.

Embodiment D110

The multi-chain chimeric polypeptide of any one of embodiments D86-D108,wherein the one or more additional target-binding domains is a solubleinterleukin or cytokine protein.

Embodiment D111

The multi-chain chimeric polypeptide of embodiment D110, wherein thesoluble interleukin, cytokine, or ligand protein is selected from thegroup consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment D112

The multi-chain chimeric polypeptide of any one of embodiments D86-D108,wherein the one or more additional target-binding domains is a solubleinterleukin or cytokine receptor.

Embodiment D113

The multi-chain chimeric polypeptide of embodiment D112, wherein thesoluble receptor is a soluble TGF-β receptor II (TGF-βRII), a solubleTGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a solubleNKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155,a soluble CD122, or a soluble CD28.

Embodiment D114

A composition comprising any of the multi-chain chimeric polypeptides ofembodiments D1-D113.

Embodiment D115

The composition of embodiment D114, wherein the composition is apharmaceutical composition.

Embodiment D116

A kit comprising at least one dose of the composition of embodiment D114or D115.

Embodiment D117

A method of stimulating an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments D1-D113 or thecomposition of embodiment D114 or D115.

Embodiment D118

The method of embodiment D117, wherein the immune cell is contacted invitro.

Embodiment D119

The method of embodiment D118, wherein the immune cell was previouslyobtained from a subject.

Embodiment D120

The method of embodiment D119, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment D121

The method of embodiment D117, wherein the immune cell is contacted invivo.

Embodiment D122

The method of any one of embodiments D117-D121, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ T cell, aCD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment D123

The method of any one of embodiments D117-D122, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment D124

The method of any one of embodiments D117-D122, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment D125

The method of any one of embodiments D117-D124, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment D126

The method of embodiment D125, wherein the subject has been identifiedor diagnosed as having age-related disease or condition.

Embodiment D127

The method of embodiment D126, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment D128

The method of embodiment D125, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment D129

The method of embodiment D128, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment D130

The method of embodiment D125, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment D131

The method of embodiment D130, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, hepatitis C virus,papillomavirus, or influenza virus.

Embodiment D132

A method of inducing or increasing proliferation of an immune cell, themethod comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments D1-D113 or thecomposition of embodiment D114 or D115.

Embodiment D133

The method of embodiment D132, wherein the immune cell is contacted invitro.

Embodiment D134

The method of embodiment D133, wherein the immune cell was previouslyobtained from a subject.

Embodiment D135

The method of embodiment D134, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment D136

The method of embodiment D132, wherein the immune cell is contacted invivo.

Embodiment D137

The method of any one of embodiments D132-D136, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, aCD4+ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment D138

The method of any one of embodiments D132-D137, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment D139

The method of any one of embodiments D132-D137, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment D140

The method of any one of embodiments D132-D139, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment D141

The method of embodiment D140, wherein the subject has been identifiedor diagnosed as having an age-related disease or condition.

Embodiment D142

The method of embodiment D141, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment D143

The method of embodiment D140, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment D144

The method of embodiment D143, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment D145

The method of embodiment D140, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment D146

The method of embodiment D140, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, hepatitis C virus,papillomavirus, or influenza virus.

Embodiment D147

A method of inducing differentiation of an immune cell into a memory ormemory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments D1-D113 or thecomposition of embodiment D114 or D115.

Embodiment D148

The method of embodiment D147, wherein the immune cell is contacted invitro.

Embodiment D149

The method of embodiment D148, wherein the immune cell was previouslyobtained from a subject.

Embodiment D150

The method of embodiment D149, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment D151

The method of embodiment D147, wherein the immune cell is contacted invivo.

Embodiment D152

The method of any one of embodiments D147-D151, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a Th17 cell, a Th22cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ Tcell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, anatural killer T cell, a mast cell, a macrophage, a neutrophil, adendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment D153

The method of any one of embodiments D147-D152, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment D154

The method of any one of embodiments D147-D152, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment D155

The method of any one of embodiments D147-D153, wherein the immune cellis contacted with anti-TF IgG antibody to create a memory or memory likeimmune cell.

Embodiment D156

The method of any one of embodiments D147-D155, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment D157

The method of embodiment D156, wherein the subject has been identifiedor diagnosed as having an age-related disease or condition.

Embodiment D158

The method of embodiment D156, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment D159

The method of embodiment D156, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment D160

The method of embodiment D159, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment D161

The method of embodiment D156, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment D162

The method of embodiment D161, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, hepatitis C virus,papillomavirus, or influenza virus.

Embodiment D163

A method of killing a cancer cell, an infected cell, or a senescent cellin a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of any of the multi-chainchimeric polypeptides of embodiments D1-D113 or the composition ofembodiment D114 or D115.

Embodiment D164

The method of embodiment D163, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment D165

The method of embodiment D164, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment D166

The method of embodiment D163, wherein the subject has been identifiedor diagnosed as having an aging-related disease or condition.

Embodiment D167

The method of embodiment D166, wherein the aging-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction

Embodiment D168

A method of treating a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anyof the multi-chain chimeric polypeptides of embodiments D1-D113 or thecomposition of embodiment D114 or D115.

Embodiment D169

The method of embodiment D168, wherein the subject has been identifiedor diagnosed as having a cancer or infectious disease.

Embodiment D170

The method of embodiment D169, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment D171

The method of embodiment D168, wherein the subject has been identifiedor diagnosed as having age-related disease or condition.

Embodiment D172

The method of embodiment D171, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment D173

The method of embodiment D168, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment D174

The method of embodiment D173, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, hepatitis C virus,papillomavirus, or influenza virus.

Embodiment D175

Nucleic acid encoding any of the multi-chain chimeric polypeptides ofany one of embodiments D1-D113.

Embodiment D176

A vector comprising the nucleic acid of embodiment D174.

Embodiment D177

The vector of embodiment D176, wherein the vector is an expressionvector.

Embodiment D178

A cell comprising the nucleic acid of embodiment D175 or the vector ofembodiment D175 or D176.

Embodiment D179

A method of producing a multi-chain chimeric polypeptide, the methodcomprising:

culturing the cell of embodiment D177 in a culture medium underconditions sufficient to result in the production of the multi-chainchimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or theculture medium.

Embodiment D180

A multi-chain chimeric polypeptide produced by the method of embodimentD179.

E. EXEMPLARY EMBODIMENTS Embodiment E1

A multi-chain chimeric polypeptide comprising:

(e) a first chimeric polypeptide comprising:

-   -   (i) a first target-binding domain;    -   (ii) a soluble tissue factor domain; and    -   (iii) a first domain of a pair of affinity domains;

(f) a second chimeric polypeptide comprising:

-   -   (i) a second domain of a pair of affinity domains; and    -   (ii) a second target-binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptideassociate through the binding of the first domain and the second domainof the pair of affinity domains; and

the first target-binding domain and the second targeting-binding domaineach independently bind specifically to: a receptor for IL-7, CD16, areceptor for IL-21, TGF-β, or a receptor for CD137L.

Embodiment E2

The multi-chain chimeric polypeptide of embodiment E1, wherein the firsttarget-binding domain and the soluble tissue factor domain directly abuteach other in the first chimeric polypeptide.

Embodiment E3

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide further comprises a linker sequence between thefirst target-binding domain and the soluble tissue factor domain in thefirst chimeric polypeptide.

Embodiment E4

The multi-chain chimeric polypeptide of any one of embodiments E1-E3,wherein the soluble tissue factor domain and the first domain of thepair of affinity domains directly abut each other in the first chimericpolypeptide.

Embodiment E5

The multi-chain chimeric polypeptide of any one of embodiments E1-E3,wherein the first chimeric polypeptide further comprises a linkersequence between the soluble tissue factor domain and the first domainof the pair of affinity domains in the first chimeric polypeptide.

Embodiment E6

The multi-chain chimeric polypeptide of any one of embodiments E1-E5,wherein the second domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide.

Embodiment E7

The multi-chain chimeric polypeptide of any one of embodiments E1-E5,wherein second chimeric polypeptide further comprises a linker sequencebetween the second domain of the pair of affinity domains and the secondtarget-binding domain in the second chimeric polypeptide.

Embodiment E8

The multi-chain chimeric polypeptide of any one of embodiments E1-E7,wherein the soluble tissue factor domain is a soluble human tissuefactor domain.

Embodiment E9

The multi-chain chimeric polypeptide of embodiment E8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 1.

Embodiment E10

The multi-chain chimeric polypeptide of embodiment E9, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 1.

Embodiment E11

The multi-chain chimeric polypeptide of embodiment E10, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 1.

Embodiment E12

The multi-chain chimeric polypeptide of any one of embodiments E8-E11,wherein the soluble human tissue factor domain does not comprise one ormore of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment E13

The multi-chain chimeric polypeptide of embodiment E12, wherein thesoluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment E14

The multi-chain chimeric polypeptide of any one of embodiments E1-E13,wherein the soluble tissue factor domain is not capable of binding toFactor VIIa.

Embodiment E15

The multi-chain chimeric polypeptide of any one of embodiments E1-E14,wherein the soluble tissue factor domain does not convert inactiveFactor X into Factor Xa.

Embodiment E16

The multi-chain chimeric polypeptide of any one of embodiments E1-E15,wherein the multi-chain chimeric polypeptide does not stimulatecoagulation in a mammal.

Embodiment E17

The multi-chain chimeric polypeptide of any one of embodiments E1-E16,wherein the first chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the first chimericpolypeptide.

Embodiment E18

The multi-chain chimeric polypeptide of any one of embodiments E1-E17,wherein the second chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the second chimericpolypeptide.

Embodiment E19

The multi-chain chimeric polypeptide of any one of embodiments E1-E18,wherein the first chimeric polypeptide and/or the second chimericpolypeptide further comprises a signal sequence at its N-terminal end.

Embodiment E20

The multi-chain chimeric polypeptide of embodiment E19, wherein thesignal sequence comprises SEQ ID NO: 31.

Embodiment E21

The multi-chain chimeric polypeptide of embodiment E20, wherein thesignal sequence is SEQ ID NO: 31.

Embodiment E22

The multi-chain chimeric polypeptide of any one of embodiments E1-E21,wherein the pair of affinity domains is a sushi domain from an alphachain of human IL-15 receptor (IL15Rα) and a soluble IL-15.

Embodiment E23

The multi-chain chimeric polypeptide of embodiment E22, wherein thesoluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment E24

The multi-chain chimeric polypeptide of embodiment E22, wherein thesoluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO:14.

Embodiment E25

The multi-chain chimeric polypeptide of embodiment E24, wherein thesoluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO:14.

Embodiment E26

The multi-chain chimeric polypeptide of embodiment E25, wherein thesoluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO:14.

Embodiment E27

The multi-chain chimeric polypeptide of embodiment E26, wherein thesoluble IL-15 comprises SEQ ID NO: 14.

Embodiment E28

The multi-chain chimeric polypeptide of any one of embodiments E22-E27,wherein the sushi domain of IL15Rα comprises a sushi domain from humanIL15Rα.

Embodiment E29

The multi-chain chimeric polypeptide of embodiment E28, wherein thesushi domain from human IL15Rα comprises a sequence that is 80%identical to SEQ ID NO: 28.

Embodiment E30

The multi-chain chimeric polypeptide of embodiment E29, wherein thesushi domain from human IL15Rα comprises a sequence that is 90%identical to SEQ ID NO: 28.

Embodiment E31

The multi-chain chimeric polypeptide of embodiment E30, wherein thesushi domain from human IL15Rα comprises a sequence that is 95%identical to SEQ ID NO: 28.

Embodiment E32

The multi-chain chimeric polypeptide of embodiment E31, wherein thesushi domain from human IL15Rα comprises SEQ ID NO: 28.

Embodiment E33

The multi-chain chimeric polypeptide of embodiment E28, wherein thesushi domain from human IL15Rα is a mature full-length IL15Rα.

Embodiment E34

The multi-chain chimeric polypeptide of any one of embodiments E1-E21,wherein the pair of affinity domains is selected from the groupconsisting of: barnase and barnstar, a PKA and an AKAP, adapter/dockingtag modules based on mutated RNase I fragments, and SNARE modules basedon interactions of the proteins syntaxin, synaptotagmin, synaptobrevin,and SNAP25.

Embodiment E35

The multi-chain chimeric polypeptide of any one of embodiments E1-E34,wherein the first target-binding domain and the second targeting-bindingdomain each independently bind specifically to a receptor for IL-7,CD16, or a receptor for IL-21.

Embodiment E36

The multi-chain chimeric polypeptide of embodiment E35, wherein thefirst target-binding domain binds specifically to a receptor IL-7 andthe second target-binding domain binds specifically to CD16 or areceptor for IL-21.

Embodiment E37

The multi-chain chimeric polypeptide of embodiment E36, wherein thefirst target-binding domain comprises a soluble IL-7 protein.

Embodiment E38

The multi-chain chimeric polypeptide of embodiment E37, wherein thesoluble IL-7 protein is a soluble human IL-7.

Embodiment E39

The multi-chain chimeric polypeptide of any one of embodiments E36-E38,wherein the second antigen-binding domain comprises an antigen-bindingdomain that binds specifically to CD16.

Embodiment E40

The multi-chain chimeric polypeptide of embodiment E39, wherein thesecond antigen-binding domain comprises an scFv that binds specificallyto CD16.

Embodiment E41

The multi-chain chimeric polypeptide of any one of embodiments E36-E38,wherein the second antigen-binding domain bind specifically to areceptor for IL-21.

Embodiment E42

The multi-chain chimeric polypeptide of embodiment E41, wherein thesecond antigen-binding domain comprises a soluble IL-21.

Embodiment E43

The multi-chain chimeric polypeptide of embodiment E42, wherein thesoluble IL-21 is a soluble human IL-21.

Embodiment E44

The multi-chain chimeric polypeptide of any one of embodiments E36-E40,wherein the second chimeric polypeptide further comprises an additionaltarget-binding domain that binds specifically to a receptor for IL-21.

Embodiment E45

The multi-chain chimeric polypeptide of embodiment E44, wherein theadditional target-binding domain comprises a soluble IL-21.

Embodiment E46

The multi-chain chimeric polypeptide of embodiment E45, wherein thesoluble IL-21 is a soluble human IL-12.

Embodiment E47

The multi-chain chimeric polypeptide of any one of embodiments E1-E34,wherein the first target-binding domain and the second targeting-bindingdomain each independently bind specifically to TGF-β, CD16, or areceptor for IL-21.

Embodiment E48

The multi-chain chimeric polypeptide of embodiment E47, wherein thefirst target-binding domain binds specifically to a TGF-β and the secondtarget-binding domain binds specifically to CD16 or a receptor of IL-21.

Embodiment E49

The multi-specific chimeric polypeptide of embodiment E48, wherein thefirst target-binding domain is a soluble TGF-β receptor.

Embodiment E50

The multi-specific chimeric polypeptide of embodiment E49, whereinsoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E51

The multi-specific chimeric polypeptide of any one of embodimentsE48-E50, wherein the second target-binding domain binds specifically toCD16.

Embodiment E52

The multi-specific chimeric polypeptide of embodiment E51, wherein thesecond antigen-binding domain comprises an antigen-binding domain thatbinds specifically to CD16.

Embodiment E53

The multi-chain chimeric polypeptide of embodiment E52, wherein thesecond antigen-binding domain comprises an scFv that binds specificallyto CD16.

Embodiment E54

The multi-chain chimeric polypeptide of any one of embodiments E48-E50,wherein the second target-binding domain binds specifically to areceptor for IL-21.

Embodiment E55

The multi-chain chimeric polypeptide of embodiment E54, wherein thesecond target-binding domain comprises a soluble IL-21.

Embodiment E56

The multi-chain chimeric polypeptide of embodiment E55, wherein thesecond target-binding domain comprises a soluble human IL-21.

Embodiment E57

The multi-chain chimeric polypeptide of any one of embodiments E48-E53,wherein the second chimeric polypeptide further comprises an additionaltarget-binding domain that binds specifically to a receptor for IL-21.

Embodiment E58

The multi-chain chimeric polypeptide of embodiment E57, wherein theadditional target-binding domain comprises a soluble IL-21.

Embodiment E59

The multi-chain chimeric polypeptide of embodiment E58, wherein thesoluble IL-21 is a soluble human IL-21.

Embodiment E60

The multi-chain chimeric polypeptide of any one of embodiments E1-E34,wherein the first target-binding domain and the second target-bindingdomain each independently bind specifically to a receptor for IL-7.

Embodiment E61

The multi-chain chimeric polypeptide of embodiment E60, wherein thefirst target-binding domain and the second target-binding domain includea soluble IL-7.

Embodiment E62

The multi-chain chimeric polypeptide of embodiment E61, wherein thesoluble IL-7 is a soluble human IL-7.

Embodiment E63

The multi-chain chimeric polypeptide of any one of embodiments E1-E34,wherein the first target-binding domain and the second target-bindingdomain each independently bind specifically to TGF-β.

Embodiment E64

The multi-specific chimeric polypeptide of embodiment E63, wherein thefirst target-binding domain and the second target-binding domain is asoluble TGF-β receptor.

Embodiment E65

The multi-specific chimeric polypeptide of embodiment E64, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E66

The multi-specific chimeric polypeptide of any one of embodimentsE1-E34, wherein the first target-binding domain and the secondtargeting-binding domain each independently bind specifically to areceptor for IL-7, a receptor for IL-21, or a receptor for CD137L.

Embodiment E67

The multi-chain chimeric polypeptide of embodiment E66, wherein thefirst target-binding domain binds specifically to a receptor for IL-7and the second target-binding domain binds specifically to a receptorfor IL-21 or a receptor for CD137L.

Embodiment E68

The multi-specific chimeric polypeptide of embodiment E67, wherein thefirst target-binding domain is a soluble IL-7.

Embodiment E69

The multi-specific chimeric polypeptide of embodiment E68, wherein thesoluble IL-7 is a soluble human IL-7.

Embodiment E70

The multi-chain chimeric polypeptide of any one of embodiments E67-E69,wherein the second target-binding domain binds specifically to areceptor for IL-21.

Embodiment E71

The multi-chain chimeric polypeptide of embodiment E70, wherein thesecond target-binding domain is a soluble IL-21.

Embodiment E72

The multi-chain chimeric polypeptide of embodiment E71, wherein thesoluble IL-21 is a soluble human IL-21.

Embodiment E73

The multi-chain chimeric polypeptide of any one of embodiments E67-E69,wherein the second antigen-binding domain binds specifically to areceptor for CD137L.

Embodiment E74

The multi-chain chimeric polypeptide of embodiment E73, wherein thesecond antigen-binding domain is a soluble CD137L.

Embodiment E75

The multi-chain chimeric polypeptide of embodiment E74, wherein thesoluble CD137L is a soluble human CD137L.

Embodiment E76

The multi-chain chimeric polypeptide of any one of embodiments E67-E72,wherein the second chimeric polypeptide further comprises an additionaltarget-binding domain that binds specifically to a receptor for CD137L.

Embodiment E77

The multi-chain chimeric polypeptide of embodiment E76, wherein theadditional target-binding domain comprises a soluble CD137L.

Embodiment E78

The multi-chain chimeric polypeptide of embodiment E77, wherein thesoluble CD137L is a soluble human CD137L.

Embodiment E79

The multi-chain chimeric polypeptide of any one of embodiments E1-E34,wherein the first target-binding domain and the second targeting-bindingdomain each independently bind specifically to a receptor for IL-7 orTGF-β.

Embodiment E80

The multi-chain chimeric polypeptide of embodiment E79, wherein thefirst target-binding domain binds specifically to a receptor IL-7 andthe second target-binding domain binds specifically to TGF-β.

Embodiment E81

The multi-chain chimeric polypeptide of embodiment E80, wherein thefirst target-binding domain comprises a soluble IL-7 protein.

Embodiment E82

The multi-chain chimeric polypeptide of embodiment E81, wherein thesoluble IL-7 protein is a soluble human IL-7.

Embodiment E83

The multi-chain chimeric polypeptide of any one of embodiments E80-E82,wherein the second antigen-binding domain comprises an antigen-bindingdomain that binds specifically to TGF-β.

Embodiment E84

The multi-specific chimeric polypeptide of embodiment E83, wherein thesecond target-binding domain is a soluble TGF-β receptor.

Embodiment E85

The multi-specific chimeric polypeptide of embodiment E84, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E86

The multi-chain chimeric polypeptide of any one of embodiments E1-E34,wherein the first target-binding domain and the second targeting-bindingdomain each independently bind specifically to TGF-β, a receptor forIL-21, or a receptor for CD137L.

Embodiment E87

The multi-chain chimeric polypeptide of embodiment E86, wherein thefirst target-binding domain binds specifically to a TGF-β and the secondtarget-binding domain binds specifically to a receptor for IL-21 or areceptor for CD137L.

Embodiment E88

The multi-specific chimeric polypeptide of embodiment E87, wherein thefirst target-binding domain is a soluble TGF-β receptor.

Embodiment E89

The multi-specific chimeric polypeptide of embodiment E88, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E90

The multi-specific chimeric polypeptide of any one of embodimentsE87-E89, wherein the second target-binding domain binds specifically toa receptor for IL-21.

Embodiment E91

The multi-chain chimeric polypeptide of embodiment E90, wherein thesecond target-binding domain comprises a soluble IL-21.

Embodiment E92

The multi-chain chimeric polypeptide of embodiment E91, wherein thesecond target-binding domain comprises a soluble human IL-21.

Embodiment E93

The multi-specific chimeric polypeptide of any one of embodimentsE87-E89, wherein the second target-binding domain binds specifically toa receptor for CD137L.

Embodiment E94

The multi-chain chimeric polypeptide of embodiment E93, wherein thesecond target-binding domain comprises a soluble CD137L.

Embodiment E95

The multi-chain chimeric polypeptide of embodiment E94, wherein thesecond target-binding domain comprises a soluble human CD137L.

Embodiment E96

The multi-chain chimeric polypeptide of any one of embodiments E87-E92,wherein the second chimeric polypeptide further comprises an additionaltarget-binding domain that binds specifically to a receptor for CD137L.

Embodiment E97

The multi-chain chimeric polypeptide of embodiment E96, wherein theadditional target-binding domain comprises a soluble CD137L.

Embodiment E98

The multi-chain chimeric polypeptide of embodiment E97, wherein thesoluble CD137L is a soluble human CD137L.

Embodiment E99

The multi-chain chimeric polypeptide of any one of embodiments E1-E34,wherein the first target-binding domain and the second targeting-bindingdomain each independently bind specifically to TGF-β or a receptor forIL-21.

Embodiment E100

The multi-chain chimeric polypeptide of embodiment E99, wherein thefirst target-binding domain binds specifically to a TGF-β and the secondtarget-binding domain binds specifically to TGF-β or a receptor forIL-21.

Embodiment E101

The multi-specific chimeric polypeptide of embodiment E100, wherein thefirst target-binding domain is a soluble TGF-β receptor.

Embodiment E102

The multi-specific chimeric polypeptide of embodiment E101, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E103

The multi-specific chimeric polypeptide of any one of embodimentsE100-E102, wherein the second target-binding domain binds specificallyto a receptor for IL-21.

Embodiment E104

The multi-chain chimeric polypeptide of embodiment E103, wherein thesecond target-binding domain comprises a soluble IL-21.

Embodiment E105

The multi-chain chimeric polypeptide of embodiment E104, wherein thesecond target-binding domain comprises a soluble human IL-21.

Embodiment E106

The multi-specific chimeric polypeptide of any one of embodimentsE100-E102, wherein the second target-binding domain binds specificallyto TGF-β.

Embodiment E107

The multi-specific chimeric polypeptide of embodiment E106, wherein thefirst target-binding domain is a soluble TGF-β receptor.

Embodiment E108

The multi-specific chimeric polypeptide of embodiment E107, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E109

The multi-specific chimeric polypeptide of any one of embodimentsE100-E105, wherein the second polypeptide further comprises anadditional target-binding domain that binds specifically to TGF-β.

Embodiment E110

The multi-specific chimeric polypeptide of embodiment E109, wherein thefirst target-binding domain is a soluble TGF-β receptor.

Embodiment E111

The multi-specific chimeric polypeptide of embodiment E110, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E112

The multi-chain chimeric polypeptide of any one of embodiments E1-E34,wherein the first target-binding domain and the second targeting-bindingdomain each independently bind specifically to TGF-β or IL-16.

Embodiment E113

The multi-chain chimeric polypeptide of embodiment E112, wherein thefirst target-binding domain binds specifically to a TGF-β and the secondtarget-binding domain binds specifically to TGF-β or IL-16.

Embodiment E114

The multi-specific chimeric polypeptide of embodiment E113, wherein thefirst target-binding domain is a soluble TGF-β receptor.

Embodiment E115

The multi-specific chimeric polypeptide of embodiment E114, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E116

The multi-specific chimeric polypeptide of any one of embodimentsE113-E115, wherein the second target-binding domain binds specificallyto IL-16.

Embodiment E117

The multi-specific chimeric polypeptide of embodiment E116, wherein thesecond antigen-binding domain comprises an antigen-binding domain thatbinds specifically to CD16.

Embodiment E118

The multi-chain chimeric polypeptide of embodiment E117, wherein thesecond antigen-binding domain comprises an scFv that binds specificallyto CD16.

Embodiment E119

The multi-specific chimeric polypeptide of any one of embodimentsE113-E115, wherein the second target-binding domain binds specificallyto TGF-β.

Embodiment E120

The multi-specific chimeric polypeptide of embodiment E119, wherein thefirst target-binding domain is a soluble TGF-β receptor.

Embodiment E121

The multi-specific chimeric polypeptide of embodiment E120, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E122

The multi-specific chimeric polypeptide of any one of embodimentsE113-E118, wherein the second chimeric polypeptide further comprises anadditional target-binding domain that binds specifically to TGF-β.

Embodiment E123

The multi-specific chimeric polypeptide of embodiment E122, wherein thefirst target-binding domain is a soluble TGF-β receptor.

Embodiment E124

The multi-specific chimeric polypeptide of embodiment E123, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E125

The multi-chain chimeric polypeptide of any one of embodiments E1-E34,wherein the first target-binding domain and the second targeting-bindingdomain each independently bind specifically to a TGF-β or a receptor forCD137L.

Embodiment E126

The multi-chain chimeric polypeptide of embodiment E125, wherein thefirst target-binding domain binds specifically to TGF-β and the secondtarget-binding domain binds specifically to a receptor for CD137L.

Embodiment E127

The multi-specific chimeric polypeptide of embodiment E126, wherein thefirst target-binding domain is a soluble TGF-β receptor.

Embodiment E128

The multi-specific chimeric polypeptide of embodiment E127, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E129

The multi-chain chimeric polypeptide of embodiment E128, wherein thesecond target-binding domain comprises a soluble CD137L protein.

Embodiment E130

The multi-chain chimeric polypeptide of embodiment E129, wherein thesoluble CD137L protein is a soluble human CD137L.

Embodiment E131

The multi-chain chimeric polypeptide of any one of embodimentsE126-E130, wherein the second chimeric polypeptide further comprises anadditional target-binding domain that binds specifically to TGF-β.

Embodiment E132

The multi-specific chimeric polypeptide of embodiment E131, wherein theadditional target-binding domain is a soluble TGF-β receptor.

Embodiment E133

The multi-specific chimeric polypeptide of embodiment E132, wherein thesoluble TGF-β receptor is a soluble TGFβRII receptor.

Embodiment E134

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 104.

Embodiment E135

The multi-chain chimeric polypeptide of embodiment E134, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 104.

Embodiment E136

The multi-chain chimeric polypeptide of embodiment E135, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 104.

Embodiment E137

The multi-chain chimeric polypeptide of embodiment E136, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 104.

Embodiment E138

The multi-chain chimeric polypeptide of embodiment E137, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 106.

Embodiment E139

The multi-chain chimeric polypeptide of any one of embodiments E1 andE134-E138, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 129.

Embodiment E140

The multi-chain chimeric polypeptide of embodiment E139, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 129.

Embodiment E141

The multi-chain chimeric polypeptide of embodiment E140, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 129.

Embodiment E142

The multi-chain chimeric polypeptide of embodiment E141, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 129.

Embodiment E143

The multi-chain chimeric polypeptide of embodiment E142, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 131.

Embodiment E144

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 133.

Embodiment E145

The multi-chain chimeric polypeptide of embodiment E144, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 133.

Embodiment E146

The multi-chain chimeric polypeptide of embodiment E145, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 133.

Embodiment E147

The multi-chain chimeric polypeptide of embodiment E146, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 133.

Embodiment E148

The multi-chain chimeric polypeptide of embodiment E147, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 135.

Embodiment E149

The multi-chain chimeric polypeptide of any one of embodiments E1 andE144-E148, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 137.

Embodiment E150

The multi-chain chimeric polypeptide of embodiment E149, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 137.

Embodiment E151

The multi-chain chimeric polypeptide of embodiment E150, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 137.

Embodiment E152

The multi-chain chimeric polypeptide of embodiment E151, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 137.

Embodiment E153

The multi-chain chimeric polypeptide of embodiment E152, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 139.

Embodiment E154

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 141.

Embodiment E155

The multi-chain chimeric polypeptide of embodiment E154, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 141.

Embodiment E156

The multi-chain chimeric polypeptide of embodiment E155, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 141.

Embodiment E157

The multi-chain chimeric polypeptide of embodiment E156, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 141.

Embodiment E158

The multi-chain chimeric polypeptide of embodiment E157, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 143.

Embodiment E159

The multi-chain chimeric polypeptide of any one of embodiments E1 andE154-E158, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 145.

Embodiment E160

The multi-chain chimeric polypeptide of embodiment E159, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 145.

Embodiment E161

The multi-chain chimeric polypeptide of embodiment E160, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 145.

Embodiment E162

The multi-chain chimeric polypeptide of embodiment E161, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 145.

Embodiment E163

The multi-chain chimeric polypeptide of embodiment E162, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 147.

Embodiment E164

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 149.

Embodiment E165

The multi-chain chimeric polypeptide of embodiment E164, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 149.

Embodiment E166

The multi-chain chimeric polypeptide of embodiment E165, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 149.

Embodiment E167

The multi-chain chimeric polypeptide of embodiment E166, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 149.

Embodiment E168

The multi-chain chimeric polypeptide of embodiment E167, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 151.

Embodiment E169

The multi-chain chimeric polypeptide of any one of embodiments E1 andE164-E168, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 153.

Embodiment E170

The multi-chain chimeric polypeptide of embodiment E169, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 153.

Embodiment E171

The multi-chain chimeric polypeptide of embodiment E170, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 153.

Embodiment E172

The multi-chain chimeric polypeptide of embodiment E171, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 153.

Embodiment E173

The multi-chain chimeric polypeptide of embodiment E172, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 155.

Embodiment E174

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 161.

Embodiment E175

The multi-chain chimeric polypeptide of embodiment E174, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 161.

Embodiment E176

The multi-chain chimeric polypeptide of embodiment E175, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 161.

Embodiment E177

The multi-chain chimeric polypeptide of embodiment E176, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 161.

Embodiment E178

The multi-chain chimeric polypeptide of embodiment E177, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 163.

Embodiment E179

The multi-chain chimeric polypeptide of any one of embodiments E1 andE174-E178, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 165.

Embodiment E180

The multi-chain chimeric polypeptide of embodiment E179, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 165.

Embodiment E181

The multi-chain chimeric polypeptide of embodiment E180, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 165.

Embodiment E182

The multi-chain chimeric polypeptide of embodiment E181, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 165.

Embodiment E183

The multi-chain chimeric polypeptide of embodiment E182, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 167.

Embodiment E184

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 173.

Embodiment E185

The multi-chain chimeric polypeptide of embodiment E184, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 173.

Embodiment E186

The multi-chain chimeric polypeptide of embodiment E185, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 173.

Embodiment E187

The multi-chain chimeric polypeptide of embodiment E186, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 173.

Embodiment E188

The multi-chain chimeric polypeptide of embodiment E187, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 175.

Embodiment E189

The multi-chain chimeric polypeptide of any one of embodiments E1 andE184-E188, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 189.

Embodiment E190

The multi-chain chimeric polypeptide of embodiment E189, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 189.

Embodiment E191

The multi-chain chimeric polypeptide of embodiment E190, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 189.

Embodiment E192

The multi-chain chimeric polypeptide of embodiment E191, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 189.

Embodiment E193

The multi-chain chimeric polypeptide of embodiment E192, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 191.

Embodiment E194

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 173.

Embodiment E195

The multi-chain chimeric polypeptide of embodiment E194, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 173.

Embodiment E196

The multi-chain chimeric polypeptide of embodiment E195, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 173.

Embodiment E197

The multi-chain chimeric polypeptide of embodiment E196, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 173.

Embodiment E198

The multi-chain chimeric polypeptide of embodiment E197, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 175.

Embodiment E199

The multi-chain chimeric polypeptide of any one of embodiments E1 andE194-E198, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 177.

Embodiment E200

The multi-chain chimeric polypeptide of embodiment E199, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 177.

Embodiment E201

The multi-chain chimeric polypeptide of embodiment E200, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 177.

Embodiment E202

The multi-chain chimeric polypeptide of embodiment E201, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 177.

Embodiment E203

The multi-chain chimeric polypeptide of embodiment E202, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 179.

Embodiment E204

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 181.

Embodiment E205

The multi-chain chimeric polypeptide of embodiment E204, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 181.

Embodiment E206

The multi-chain chimeric polypeptide of embodiment E205, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 181.

Embodiment E207

The multi-chain chimeric polypeptide of embodiment E206, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 181.

Embodiment E208

The multi-chain chimeric polypeptide of embodiment E207, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 183.

Embodiment E209

The multi-chain chimeric polypeptide of any one of embodiments E1 andE204-E208, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 185.

Embodiment E210

The multi-chain chimeric polypeptide of embodiment E209, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 185.

Embodiment E211

The multi-chain chimeric polypeptide of embodiment E210, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 185.

Embodiment E212

The multi-chain chimeric polypeptide of embodiment E211, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 185.

Embodiment E213

The multi-chain chimeric polypeptide of embodiment E212, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 187.

Embodiment E214

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 193.

Embodiment E215

The multi-chain chimeric polypeptide of embodiment E214, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 193.

Embodiment E216

The multi-chain chimeric polypeptide of embodiment E215, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 193.

Embodiment E217

The multi-chain chimeric polypeptide of embodiment E216, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 193.

Embodiment E218

The multi-chain chimeric polypeptide of embodiment E217, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 195.

Embodiment E219

The multi-chain chimeric polypeptide of any one of embodiments E1 andE214-E218, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 197.

Embodiment E220

The multi-chain chimeric polypeptide of embodiment E219, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 197.

Embodiment E221

The multi-chain chimeric polypeptide of embodiment E220, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 197.

Embodiment E222

The multi-chain chimeric polypeptide of embodiment E221, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 197.

Embodiment E223

The multi-chain chimeric polypeptide of embodiment E222, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 199.

Embodiment E224

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 201.

Embodiment E225

The multi-chain chimeric polypeptide of embodiment E224, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 201.

Embodiment E226

The multi-chain chimeric polypeptide of embodiment E225, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 201.

Embodiment E227

The multi-chain chimeric polypeptide of embodiment E226, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 201.

Embodiment E228

The multi-chain chimeric polypeptide of embodiment E227, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 203.

Embodiment E229

The multi-chain chimeric polypeptide of any one of embodiments E1 andE224-E228, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 205.

Embodiment E230

The multi-chain chimeric polypeptide of embodiment E229, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 205.

Embodiment E231

The multi-chain chimeric polypeptide of embodiment E230, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 205.

Embodiment E232

The multi-chain chimeric polypeptide of embodiment E231, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 205.

Embodiment E233

The multi-chain chimeric polypeptide of embodiment E232, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 207.

Embodiment E234

The multi-chain chimeric polypeptide of embodiment E1, wherein the firstchimeric polypeptide comprises a sequence that is at least 80% identicalto SEQ ID NO: 209.

Embodiment E235

The multi-chain chimeric polypeptide of embodiment E234, wherein thefirst chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 209.

Embodiment E236

The multi-chain chimeric polypeptide of embodiment E235, wherein thefirst chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 209.

Embodiment E237

The multi-chain chimeric polypeptide of embodiment E236, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 209.

Embodiment E238

The multi-chain chimeric polypeptide of embodiment E237, wherein thefirst chimeric polypeptide comprises SEQ ID NO: 211.

Embodiment E239

The multi-chain chimeric polypeptide of any one of embodiments E1 andE234-E238, wherein the second chimeric polypeptide comprises a sequencethat is at least 80% identical to SEQ ID NO: 213.

Embodiment E240

The multi-chain chimeric polypeptide of embodiment E239, wherein thesecond chimeric polypeptide comprises a sequence that is at least 90%identical to SEQ ID NO: 213.

Embodiment E241

The multi-chain chimeric polypeptide of embodiment E240, wherein thesecond chimeric polypeptide comprises a sequence that is at least 95%identical to SEQ ID NO: 213.

Embodiment E242

The multi-chain chimeric polypeptide of embodiment E241, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 213.

Embodiment E243

The multi-chain chimeric polypeptide of embodiment E242, wherein thesecond chimeric polypeptide comprises SEQ ID NO: 215.

Embodiment E244

The multi-chain chimeric polypeptide of any one of embodiments E1-E133,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domain(s), where at least one of the one ormore additional antigen-binding domain(s) is positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains.

Embodiment E245

The multi-chain chimeric polypeptide of embodiment E244, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe soluble tissue factor domain and the at least one of the one or moreadditional antigen-binding domain(s), and/or a linker sequence betweenthe at least one of the one or more additional antigen-binding domain(s)and the first domain of the pair of affinity domains.

Embodiment E246

The multi-chain chimeric polypeptide of any one of embodiments E1-E133,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domains at the N-terminal and/or C-terminalend of the first chimeric polypeptide.

Embodiment E247

The multi-chain chimeric polypeptide of embodiment E246, wherein atleast one of the one or more additional target-binding domains directlyabuts the first domain of the pair of affinity domains in the firstchimeric polypeptide.

Embodiment E248

The multi-chain chimeric polypeptide of embodiment E246, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first domain of the pair of affinity domains.

Embodiment E249

The multi-chain chimeric polypeptide of embodiment E246, wherein the atleast one of the one or more additional target-binding domains directlyabuts the first target-binding domain in the first chimeric polypeptide.

Embodiment E250

The multi-chain chimeric polypeptide of embodiment E246, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first target-binding domain.

Embodiment E251

The multi-chain chimeric polypeptide of embodiment E246, wherein atleast one of the one or more additional target-binding domains isdisposed at the N- and/or C-terminus of the first chimeric polypeptide,and at least one of the one or more additional target-binding domains ispositioned between the soluble tissue factor domain and the first domainof the pair of affinity domains in the first chimeric polypeptide.

Embodiment E252

The multi-chain chimeric polypeptide of embodiment E251, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the N-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment E253

The multi-chain chimeric polypeptide of embodiment E251, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment E254

The multi-chain chimeric polypeptide of embodiment E251, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the C-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment E255

The multi-chain chimeric polypeptide of embodiment E251, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment E256

The multi-chain chimeric polypeptide of embodiment E251, wherein the atleast one of the one or more additional target-binding domainspositioned between the soluble tissue factor domain and the first domainof the pair of affinity domains, directly abuts the soluble tissuefactor domain and/or the first domain of the pair of affinity domains.

Embodiment E257

The multi-chain chimeric polypeptide of embodiment E251, wherein thefirst chimeric polypeptide further comprises a linker sequence disposed(i) between the soluble tissue factor domain and the at least one of theone or more additional target-binding domains positioned between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains, and/or (ii) between the first domain of the pair ofaffinity domains and the at least one of the one or more additionaltarget-binding domains positioned between the soluble tissue factordomain and the first domain of the pair of affinity domains.

Embodiment E258

The multi-chain chimeric polypeptide of any one of embodiments E44-E46,E57-E59, E76-E78, E96-E98, E109-E111, E122-E124, and E131-E133, whereinthe second chimeric polypeptide further comprises the additionaltarget-binding domain at the N-terminal end or the C-terminal end of thesecond chimeric polypeptide.

Embodiment E259

The multi-chain chimeric polypeptide of embodiment E258, wherein theadditional target-binding domain directly abuts the second domain of thepair of affinity domains in the second chimeric polypeptide.

Embodiment E260

The multi-chain chimeric polypeptide of embodiment E258, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenthe additional target-binding domain and the second domain of the pairof affinity domains in the second chimeric polypeptide.

Embodiment E261

The multi-chain chimeric polypeptide of embodiment E258, wherein theadditional target-binding domain directly abuts the secondtarget-binding domain in the second chimeric polypeptide.

Embodiment E262

The multi-chain chimeric polypeptide of embodiment E258, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenthe additional target-binding domain and the second target-bindingdomain in the second chimeric polypeptide.

Embodiment E263

A composition comprising any of the multi-chain chimeric polypeptides ofembodiments E1-E262.

Embodiment E264

The composition of embodiment E263, wherein the composition is apharmaceutical composition.

Embodiment E265

A kit comprising at least one dose of the composition of embodiment E263or E264.

Embodiment E266

A method of stimulating an immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments E1-E262 or thecomposition of embodiment E263 or E264.

Embodiment E267

The method of embodiment E266, wherein the immune cell is contacted invitro.

Embodiment E268

The method of embodiment E267, wherein the immune cell was previouslyobtained from a subject.

Embodiment E269

The method of embodiment E268, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment E270

The method of embodiment E266, wherein the immune cell is contacted invivo.

Embodiment E271

The method of any one of embodiments E266-E270, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8⁺ T cell, aCD4⁺ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment E272

The method of any one of embodiments E266-E271, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment E273

The method of any one of embodiments E266-E271, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment E274

The method of any one of embodiments E266-E273, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment E275

The method of embodiment E274, wherein the subject has been identifiedor diagnosed as having age-related disease or condition.

Embodiment E276

The method of embodiment E275, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment E277

The method of embodiment E274, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment E278

The method of embodiment E277, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment E279

The method of embodiment E274, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment E280

The method of embodiment E279, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment E281

A method of inducing or increasing proliferation of an immune cell, themethod comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments E1-E262 or thecomposition of embodiment E263 or E264.

Embodiment E282

The method of embodiment E281, wherein the immune cell is contacted invitro.

Embodiment E283

The method of embodiment E282, wherein the immune cell was previouslyobtained from a subject.

Embodiment E284

The method of embodiment E283, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment E285

The method of embodiment E281, wherein the immune cell is contacted invivo.

Embodiment E286

The method of any one of embodiments E281-E285, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a memory T cell, aTh17 cell, a Th22 cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell,γδ T cell, an αβ T cell, a tumor-infiltrating T cell, a CD8+ T cell, aCD4+ T cell, a natural killer T cell, a mast cell, a macrophage, aneutrophil, a dendritic cell, a basophil, an eosinophil, and a naturalkiller cell.

Embodiment E287

The method of any one of embodiments E281-E286, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment E288

The method of any one of embodiments E281-E286, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment E289

The method of any one of embodiments E281-E288, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment E290

The method of embodiment E289, wherein the subject has been identifiedor diagnosed as having an age-related disease or condition.

Embodiment E291

The method of embodiment E290, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment E292

The method of embodiment E289, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment E293

The method of embodiment E292, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment E294

The method of embodiment E289, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment E295

The method of embodiment E289, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment E296

A method of inducing differentiation of an immune cell into a memory ormemory-like immune cell, the method comprising:

contacting an immune cell with an effective amount of any of themulti-chain chimeric polypeptides of embodiments E1-E262 or thecomposition of embodiment E263 or E264.

Embodiment E297

The method of embodiment E296, wherein the immune cell is contacted invitro.

Embodiment E298

The method of embodiment E297, wherein the immune cell was previouslyobtained from a subject.

Embodiment E299

The method of embodiment E298, wherein the method further comprisesobtaining the immune cell from the subject prior to the contacting step.

Embodiment E300

The method of embodiment E296, wherein the immune cell is contacted invivo.

Embodiment E301

The method of any one of embodiments E296-E300, wherein the immune cellis selected from the group consisting of: an immature thymocyte, aperipheral blood lymphocyte, a naïve T cell, a pluripotent Th cellprecursor, a lymphoid progenitor cell, a Treg cell, a Th17 cell, a Th22cell, a Th9 cell, a Th2 cell, a Th1 cell, a Th3 cell, γδ T cell, an αβ Tcell, a tumor-infiltrating T cell, a CD8+ T cell, a CD4+ T cell, anatural killer T cell, a mast cell, a macrophage, a neutrophil, adendritic cell, a basophil, an eosinophil, and a natural killer cell.

Embodiment E302

The method of any one of embodiments E296-E301, wherein the immune cellhas previously been genetically modified to express a chimeric antigenreceptor or a recombinant T-cell receptor.

Embodiment E303

The method of any one of embodiments E296-E301, wherein the methodfurther comprises, after the contacting step, introducing into theimmune cell a nucleic acid encoding a chimeric antigen-receptor or arecombinant T-cell receptor.

Embodiment E304

The method of any one of embodiments E296-E303, wherein the methodfurther comprises administering the immune cell to a subject in needthereof.

Embodiment E305

The method of embodiment E304, wherein the subject has been identifiedor diagnosed as having an age-related disease or condition.

Embodiment E306

The method of embodiment E305, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment E307

The method of embodiment E304, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment E308

The method of embodiment E307, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment E309

The method of embodiment E304, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment E310

The method of embodiment E309, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment E311

A method of killing a cancer cell, an infected cell, or a senescent cellin a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of any of the multi-chainchimeric polypeptides of embodiments E1-E262 or the composition ofembodiment E263 or E264.

Embodiment E312

The method of embodiment E311, wherein the subject has been identifiedor diagnosed as having a cancer.

Embodiment E313

The method of embodiment E312, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment E314

The method of embodiment E311, wherein the subject has been identifiedor diagnosed as having an aging-related disease or condition.

Embodiment E315

The method of embodiment E314, wherein the aging-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment E316

A method of treating a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of anyof the multi-chain chimeric polypeptides of embodiments E1-E262 or thecomposition of embodiment E263 or E264.

Embodiment E317

The method of embodiment E316, wherein the subject has been identifiedor diagnosed as having a cancer or infectious disease.

Embodiment E318

The method of embodiment E317, wherein the cancer is selected from thegroup consisting of: solid tumor, hematological tumor, sarcoma,osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma,Ewing sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-celllymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chroniclymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), acute lymphocytic leukemia (ALL),myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma,retinoblastoma, stomach cancer, urothelial carcinoma, lung cancer, renalcell carcinoma, gastric and esophageal cancer, pancreatic cancer,prostate cancer, breast cancer, colorectal cancer, ovarian cancer,non-small cell lung carcinoma, squamous cell head and neck carcinoma,endometrial cancer, cervical cancer, liver cancer, and hepatocellularcarcinoma.

Embodiment E319

The method of embodiment E316, wherein the subject has been identifiedor diagnosed as having age-related disease or condition.

Embodiment E320

The method of embodiment E319, wherein the age-related disease orcondition is selected from the group consisting of: Alzheimer's disease,aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma,hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease,intervertebral disc degeneration, macular degeneration, osteoarthritis,type 2 diabetes mellitus, adipose atrophy, lipodystrophy,atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidneytransplant failure, liver fibrosis, loss of bone mass, myocardialinfarction, sarcopenia, wound healing, alopecia, cardiomyocytehypertrophy, osteoarthritis, Parkinson's disease, age-associated loss oflung tissue elasticity, macular degeneration, cachexia,glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophiclateral sclerosis, Huntington's disease, spinocerebellar ataxia,multiple sclerosis, and renal dysfunction.

Embodiment E321

The method of embodiment E316, wherein the subject has been diagnosed oridentified as having an infectious disease.

Embodiment E322

The method of embodiment E321, wherein the infectious disease isinfection with human immunodeficiency virus, cytomegalovirus,adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplexvirus, hepatitis B virus, hepatitis A virus, and hepatitis C virus,papillomavirus, and influenza virus.

Embodiment E323

Nucleic acid encoding any of the multi-chain chimeric polypeptides ofany one of embodiments E1-E262.

Embodiment E324

A vector comprising the nucleic acid of embodiment E323.

Embodiment E325

The vector of embodiment E324, wherein the vector is an expressionvector.

Embodiment E326

A cell comprising the nucleic acid of embodiment E323 or the vector ofembodiment E324 or E325.

Embodiment E327

A method of producing a multi-chain chimeric polypeptide, the methodcomprising:

culturing the cell of embodiment E326 in a culture medium underconditions sufficient to result in the production of the multi-chainchimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or theculture medium.

Embodiment E328

A multi-chain chimeric polypeptide produced by the method of embodimentE327.

Embodiment E329

The multi-chain chimeric polypeptide of embodiment E8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 3.

Embodiment E330

The multi-chain chimeric polypeptide of embodiment E329, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 3.

Embodiment E331

The multi-chain chimeric polypeptide of embodiment E330, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 3.

Embodiment E332

The multi-chain chimeric polypeptide of embodiment E331, wherein thesoluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 3.

Embodiment E333

The multi-chain chimeric polypeptide of embodiment E8, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 4.

Embodiment E334

The multi-chain chimeric polypeptide of embodiment E333, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 4.

Embodiment E335

The multi-chain chimeric polypeptide of embodiment E334, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 4.

Embodiment E336

The multi-chain chimeric polypeptide of embodiment E335, wherein thesoluble human tissue factor domain comprises a sequence that is 100%identical to SEQ ID NO: 4.

F. EXEMPLARY EMBODIMENTS Embodiment F1

A multi-chain chimeric polypeptide comprising:

(a) a first chimeric polypeptide comprising:

-   -   (i) a first target-binding domain;    -   (ii) a linker domain; and    -   (iii) a first domain of a pair of affinity domains;

(b) a second chimeric polypeptide comprising:

-   -   (i) a second domain of a pair of affinity domains; and    -   (ii) a second target-binding domain,    -   wherein the first chimeric polypeptide and the second chimeric        polypeptide associate through the binding of the first domain        and the second domain of the pair of affinity domains.

Embodiment F2

The multi-chain chimeric polypeptide of embodiment F1, wherein the firsttarget-binding domain and the linker domain directly abut each other inthe first chimeric polypeptide.

Embodiment F3

The multi-chain chimeric polypeptide of embodiment F1, wherein the firstchimeric polypeptide further comprises a linker sequence between thefirst target-binding domain and the linker domain in the first chimericpolypeptide.

Embodiment F4

The multi-chain chimeric polypeptide of any one of embodiments F1-F3,wherein the linker domain and the first domain of the pair of affinitydomains directly abut each other in the first chimeric polypeptide.

Embodiment F5

The multi-chain chimeric polypeptide of any one of embodiments F1-F3,wherein the first chimeric polypeptide further comprises a linkersequence between the linker domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.

Embodiment F6

The multi-chain chimeric polypeptide of any one of embodiments F1-F5,wherein the second domain of the pair of affinity domains and the secondtarget-binding domain directly abut each other in the second chimericpolypeptide.

Embodiment F7

The multi-chain chimeric polypeptide of any one of embodiments F1-F5,wherein the second chimeric polypeptide further comprises a linkersequence between the second domain of the pair of affinity domains andthe second target-binding domain in the second chimeric polypeptide.

Embodiment F8

The multi-chain chimeric polypeptide of any one of embodiments F1-F7,wherein the first target-binding domain and the second target-bindingdomain bind specifically to the same antigen.

Embodiment F9

The multi-chain chimeric polypeptide of embodiment F8, wherein the firsttarget-binding domain and the second target-binding domain bindspecifically to the same epitope.

Embodiment F10

The multi-chain chimeric polypeptide of embodiment F9, wherein the firsttarget-binding domain and the second target-binding domain comprise thesame amino acid sequence.

Embodiment F11

The multi-chain chimeric polypeptide of any one of embodiments F1-F7,wherein the first target-binding domain and the second target-bindingdomain bind specifically to different antigens.

Embodiment F12

The multi-chain chimeric polypeptide of any one of embodiments F1-F11,wherein one or both of the first target-binding domain and the secondtarget-binding domain is an antigen-binding domain.

Embodiment F13

The multi-chain chimeric polypeptide of embodiment F12, wherein thefirst target-binding domain and the second target-binding domain areeach antigen-binding domains.

Embodiment F14

The multi-chain chimeric polypeptide of embodiment F12 or F13, whereinthe antigen-binding domain comprises a scFv or a single domain antibody.

Embodiment F15

The multi-chain chimeric polypeptide of any one of embodiments F1-F14,wherein one or both of the first target-binding domain and the secondtarget-binding domain bind specifically to a target selected from thegroup consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123,IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4,MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, CD80,CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA,CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET,EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin,CEACAM5, a UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122,CD155, PDGF-DD, a ligand of TGF-β receptor II (TGF-βRII), a ligand ofTGF-βRIII, a ligand of DNAM-1, a ligand of NKp46, a ligand of NKp44, aligand of NKG2D, a ligand of NKp30, a ligand for a scMHCI, a ligand fora scMHCII, a ligand for a scTCR, a receptor for IL-1, a receptor forIL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, areceptor for IL-10, a receptor for IL-12, a receptor for IL-15, areceptor for IL-17, a receptor for IL-18, a receptor for IL-21, areceptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptorfor stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor forMICA, a receptor for MICB, a receptor for a ULP16-binding protein, areceptor for CD155, a receptor for CD122, and a receptor for CD28.

Embodiment F16

The multi-chain chimeric polypeptide of any one of embodiments F1-F14,wherein one or both of the first target-binding domain and the secondtarget-binding domain is a soluble interleukin or cytokine protein.

Embodiment F17

The multi-chain chimeric polypeptide of embodiment F16, wherein thesoluble interleukin, cytokine, or ligand protein is selected from thegroup consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15,IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

Embodiment F18

The multi-chain chimeric polypeptide of any one of embodiments F1-F14,wherein one or both of the first target-binding domain and the secondtarget-binding domain is a soluble interleukin or cytokine receptor.

Embodiment F19

The multi-chain chimeric polypeptide of embodiment F18, wherein thesoluble receptor is a soluble TGF-β receptor II (TGF-β RII), a solubleTGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a solubleNKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155,or a soluble CD28.

Embodiment F20

The multi-chain chimeric polypeptide of any one of embodiments F1-F19,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domain(s), where at least one of the one ormore target-binding domain(s) is positioned between the linker domainand the first domain of the pair of affinity domains.

Embodiment F21

The multi-chain chimeric polypeptide of embodiment F20, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe linker domain and the at least one of the one or more targetantigen-binding domain(s), and/or a linker sequence between the at leastone of the one or more target antigen-binding domain(s) and the firstdomain of the pair of affinity domains.

Embodiment F22

The multi-chain chimeric polypeptide of any one of embodiments F1-F19,wherein the first chimeric polypeptide further comprises one or moreadditional target-binding domains at the N-terminal and/or C-terminalend of the first chimeric polypeptide.

Embodiment F23

The multi-chain chimeric polypeptide of embodiment F22, wherein at leastone of the one or more additional target-binding domains directly abutsthe first domain of the pair of affinity domains in the first chimericpolypeptide.

Embodiment F24

The multi-chain chimeric polypeptide of embodiment F22, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first domain of the pair of affinity domains.

Embodiment F25

The multi-chain chimeric polypeptide of embodiment F22, wherein the atleast one of the one or more additional target-binding domains directlyabuts the first target-binding domain in the first chimeric polypeptide.

Embodiment F26

The multi-chain chimeric polypeptide of embodiment F22, wherein thefirst chimeric polypeptide further comprises a linker sequence betweenthe at least one of the one or more additional target-binding domainsand the first target-binding domain.

Embodiment F27

The multi-chain chimeric polypeptide of embodiment F22, wherein at leastone of the one or more additional target-binding domains is disposed atthe N- and/or C-terminus of the first chimeric polypeptide, and at leastone of the one or more additional target-binding domains is positionedbetween the linker domain and the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment F28

The multi-chain chimeric polypeptide of embodiment F27, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the N-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment F29

The multi-chain chimeric polypeptide of embodiment F27, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment F30

The multi-chain chimeric polypeptide of embodiment F27, wherein the atleast one additional target-binding domain of the one or more additionaltarget-binding domains disposed at the C-terminus directly abuts thefirst target-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment F31

The multi-chain chimeric polypeptide of embodiment F27, wherein thefirst chimeric polypeptide further comprises a linker sequence disposedbetween the at least one additional target-binding domain and the firsttarget-binding domain or the first domain of the pair of affinitydomains in the first chimeric polypeptide.

Embodiment F32

The multi-chain chimeric polypeptide of embodiment F27, wherein the atleast one of the one or more additional target-binding domainspositioned between the linker domain and the first domain of the pair ofaffinity domains, directly abuts the linker domain and/or the firstdomain of the pair of affinity domains.

Embodiment F33

The multi-chain chimeric polypeptide of embodiment F27, wherein thefirst chimeric polypeptide further comprises a linker sequence disposed(i) between the linker domain and the at least one of the one or moreadditional target-binding domains positioned between the linker domainand the first domain of the pair of affinity domains, and/or (ii)between the first domain of the pair of affinity domains and the atleast one of the one or more additional target-binding domainspositioned between the linker domain and the first domain of the pair ofaffinity domains.

Embodiment F34

The multi-chain chimeric polypeptide of any one of embodiments F1-F33,wherein the second chimeric polypeptide further comprises one or moreadditional target-binding domains at the N-terminal end and/or theC-terminal end of the second chimeric polypeptide.

Embodiment F35

The multi-chain chimeric polypeptide of embodiment F34, wherein at leastone of the one or more additional target-binding domains directly abutsthe second domain of the pair of affinity domains in the second chimericpolypeptide.

Embodiment F36

The multi-chain chimeric polypeptide of embodiment F34, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second domain of the pair of affinity domains in the second chimericpolypeptide.

Embodiment F37

The multi-chain chimeric polypeptide of embodiment F34, wherein at leastone of the one or more additional target-binding domains directly abutsthe second target-binding domain in the second chimeric polypeptide.

Embodiment F38

The multi-chain chimeric polypeptide of embodiment F34, wherein thesecond chimeric polypeptide further comprises a linker sequence betweenat least one of the one or more additional target-binding domains andthe second target-binding domain in the second chimeric polypeptide.

Embodiment F39

The multi-chain chimeric polypeptide of any one of embodiments F20-F38,wherein two or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains bind specifically to the same antigen.

Embodiment F40

The multi-chain chimeric polypeptide of embodiment F39, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to the same epitope.

Embodiment F41

The multi-chain chimeric polypeptide of embodiment F40, wherein two ormore of the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains comprisethe same amino acid sequence.

Embodiment F42

The multi-chain chimeric polypeptide of embodiment F39, wherein thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains each bind specifically tothe same antigen.

Embodiment F43

The multi-chain chimeric polypeptide of embodiment F42, wherein thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains each bind specifically tothe same epitope.

Embodiment F44

The multi-chain chimeric polypeptide of embodiment F43, wherein thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains each comprise the sameamino acid sequence.

Embodiment F45

The multi-chain chimeric polypeptide of any one of embodiments F20-F38,wherein the first target-binding domain, the second target-bindingdomain, and the one or more additional target-binding domains bindspecifically to different antigens.

Embodiment F46

The multi-chain chimeric polypeptide of any one of embodiments F20-F45,wherein one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more target-binding domains is anantigen-binding domain.

Embodiment F47

The multi-chain chimeric polypeptide of embodiment F46, wherein thefirst target-binding domain, the second target-binding domain, and theone or more additional target-binding domains are each anantigen-binding domain.

Embodiment F48

The multi-chain chimeric polypeptide of embodiment F46 or F47, whereinthe antigen-binding domain comprises a scFv.

Embodiment F49

The multi-chain chimeric polypeptide of any one of embodiments F20-F48,wherein one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more target-binding domains bindspecifically to a target selected from the group consisting of: CD16a,CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R,IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8,TNFα, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM,ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70,OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3,PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, a UL16-bindingprotein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, a ligandof TGF-β receptor II (TGF-βRII), a ligand of TGF-βRIII, a ligand ofDNAM-1, a ligand of NKp46, a ligand of NKp44, a ligand of NKG2D, aligand of NKp30, a ligand for a scMHCI, a ligand for a scMHCII, a ligandfor a scTCR, a receptor for IL-1, a receptor for IL-2, a receptor forIL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, areceptor for IL-12, a receptor for IL-15, a receptor for IL-17, areceptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, areceptor for stem cell factor (SCF), a receptor for stem cell-liketyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor forMICB, a receptor for a ULP16-binding protein, a receptor for CD155, areceptor for CD122, and a receptor for CD3, and a receptor for CD28.

Embodiment F50

The multi-chain chimeric polypeptide of any one of embodiments F20-F48,wherein one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains is a soluble interleukin or cytokine protein.

Embodiment F52

The multi-chain chimeric polypeptide of any one of embodiments F20-F48,wherein one or more of the first target-binding domain, the secondtarget-binding domain, and the one or more additional target-bindingdomains is a soluble interleukin or cytokine receptor.

Embodiment F53

The multi-chain chimeric polypeptide of embodiment F52, wherein thesoluble receptor is a soluble TGF-β receptor II (TGF-β RII), a solubleTGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a solubleNKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155,a soluble CD122, a soluble CD3, or a soluble CD28.

Embodiment F54

The multi-chain chimeric polypeptide of any one of embodiments F1-F53,wherein the first chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the first chimericpolypeptide.

Embodiment F55

The multi-chain chimeric polypeptide of any one of embodiments F1-F53,wherein the second chimeric polypeptide further comprises a peptide tagat the N-terminal end or the C-terminal end of the second chimericpolypeptide.

Embodiment F56

The multi-chain chimeric polypeptide of any one of embodiments F1-F55,wherein the linker domain is a soluble tissue factor domain.

Embodiment F57

The multi-chain chimeric polypeptide of embodiment F56, wherein thesoluble tissue factor domain is a soluble human tissue factor domain.

Embodiment F58

The multi-chain chimeric polypeptide of embodiment F57, wherein thesoluble human tissue factor domain comprises a sequence that is at least80% identical to SEQ ID NO: 1.

Embodiment F59

The multi-chain chimeric polypeptide of embodiment F58, wherein thesoluble human tissue factor domain comprises a sequence that is at least90% identical to SEQ ID NO: 1.

Embodiment F60

The multi-chain chimeric polypeptide of embodiment F59, wherein thesoluble human tissue factor domain comprises a sequence that is at least95% identical to SEQ ID NO: 1.

Embodiment F61

The multi-chain chimeric polypeptide of any one of embodiments F57-F60,wherein the soluble human tissue factor domain does not comprise one ormore of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment F62

The multi-chain chimeric polypeptide of embodiment F61, wherein thesoluble human tissue factor domain does not comprise any of:

a lysine at an amino acid position that corresponds to amino acidposition 20 of mature wildtype human tissue factor protein;

an isoleucine at an amino acid position that corresponds to amino acidposition 22 of mature wildtype human tissue factor protein;

a tryptophan at an amino acid position that corresponds to amino acidposition 45 of mature wildtype human tissue factor protein;

an aspartic acid at an amino acid position that corresponds to aminoacid position 58 of mature wildtype human tissue factor protein;

a tyrosine at an amino acid position that corresponds to amino acidposition 94 of mature wildtype human tissue factor protein;

an arginine at an amino acid position that corresponds to amino acidposition 135 of mature wildtype human tissue factor protein; and

a phenylalanine at an amino acid position that corresponds to amino acidposition 140 of mature wildtype human tissue factor protein.

Embodiment F63

The multi-chain chimeric polypeptide of any one of embodiments F56-F62,wherein the soluble tissue factor domain is not capable of binding toFactor VIIa.

Embodiment F64

The multi-chain chimeric polypeptide of any one of embodiments F56-F63,wherein the soluble tissue factor domain does not convert inactiveFactor X into Factor Xa.

Embodiment F65

The multi-chain chimeric polypeptide of any one of embodiments F56-F64,wherein the multi-chain chimeric polypeptide does not stimulate bloodcoagulation in a mammal.

Embodiment F66

The multi-chain chimeric polypeptide of any one of embodiments F1-F55,wherein the linker domain is selected from the group consisting of: akappa chain and a lambda chain.

Embodiment F67

The multi-chain chimeric polypeptide of any one of embodiments F1-F66,wherein the pair of affinity domains is a sushi domain from an alphachain of human IL-15 receptor (IL15Rα) and a soluble IL-15.

Embodiment F68

The multi-chain chimeric polypeptide of embodiment F67, wherein thesoluble IL15 has a D8N or D8A amino acid substitution.

Embodiment F69

The multi-chain chimeric polypeptide of embodiment F67 or F68, whereinthe human IL15Rα is a mature full-length IL15Rα.

Embodiment F70

The multi-chain chimeric polypeptide of any one of embodiments F1-F66,wherein the pair of affinity domains is selected from the groupconsisting of: barnase and barnstar, a PKA and an AKAP, adapter/dockingtag modules based on mutated RNase I fragments, and SNARE modules basedon interactions of the proteins syntaxin, synaptotagmin, synaptobrevin,and SNAP25.

Embodiment F71

The multi-chain chimeric polypeptide of any one of embodiments F1-F70,wherein the first chimeric polypeptide and/or the second chimericpolypeptide further comprises a signal sequence at its N-terminal end.

Embodiment F72

The multi-chain chimeric polypeptide of any one of embodiments F1-F70,wherein the first chimeric polypeptide and/or the second chimericpolypeptide lacks a signal sequence at its N-terminal end.

Embodiment F73

A composition comprising any of the multi-chain chimeric polypeptides ofembodiments F1-F72.

Embodiment F74

The composition of embodiment F73, wherein the composition is apharmaceutical composition.

Embodiment F75

A kit comprising at least one dose of the composition of embodiment F73or F74.

Embodiment F76

Nucleic acid encoding any of the multi-chain chimeric polypeptides ofany one of embodiments F1-F72.

Embodiment F77

A vector comprising the nucleic acid of embodiment F76.

Embodiment F78

The vector of embodiment F77, wherein the vector is an expressionvector.

Embodiment F79

A cell comprising the nucleic acid of embodiment F76 or the vector ofembodiment F77 or F78.

Embodiment F80

A method of producing a multi-chain chimeric polypeptide, the methodcomprising:

culturing the cell of embodiment F79 in a culture medium underconditions sufficient to result in the production of the multi-chainchimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cell and/or theculture medium.

Embodiment F81

A multi-chain chimeric polypeptide produced by the method of embodimentF80.

What is claimed is:
 1. A multi-chain chimeric polypeptide comprising:(a) a first chimeric polypeptide comprising: (i) a first target-bindingdomain; (ii) a soluble tissue factor domain comprising a sequence thatis at least 90% identical to SEQ ID NO: 1; and (iii) a first domain of apair of affinity domains comprising a sequence that is at least 90%identical to SEQ ID NO: 14; and (b) a second chimeric polypeptidecomprising: (i) a second domain of a pair of affinity domains comprisinga sequence that is at least 90% identical to SEQ ID NO: 28; and (ii) asecond target-binding domain, wherein: the first chimeric polypeptideand the second chimeric polypeptide associate through the binding of thefirst domain and the second domain of the pair of affinity domains; andthe first target-binding domain and the second target-binding domaineach comprise a soluble TGF-β receptor II (TGF-βRII) and each comprise asequence that is at least 90% identical to SEQ ID NO:
 85. 2. Themulti-chain chimeric polypeptide of claim 1, wherein the first target-binding domain and the soluble tissue factor domain directly abut eachother in the first chimeric polypeptide.
 3. The multi-chain chimericpolypeptide of claim 1, wherein the first chimeric polypeptide furthercomprises a linker sequence between the first target-binding domain andthe soluble tissue factor domain in the first chimeric polypeptide. 4.The multi-chain chimeric polypeptide of claim 1, wherein the solubletissue factor domain and the first domain of the pair of affinitydomains directly abut each other in the first chimeric polypeptide. 5.The multi-chain chimeric polypeptide of claim 1, wherein the firstchimeric polypeptide further comprises a linker sequence between thesoluble tissue factor domain and the first domain of the pair ofaffinity domains in the first chimeric polypeptide.
 6. The multi-chainchimeric polypeptide of claim 1, wherein the second domain of the pairof affinity domains and the second target-binding domain directly abuteach other in the second chimeric polypeptide.
 7. The multi-chainchimeric polypeptide of claim 1, wherein the second chimeric polypeptidefurther comprises a linker sequence between the second domain of thepair of affinity domains and the second target-binding domain in thesecond chimeric polypeptide.
 8. The multi-chain chimeric polypeptide ofclaim 1, wherein the first chimeric polypeptide further comprises one ormore additional target-binding domain(s).
 9. The multi-chain chimericpolypeptide of claim 1, wherein the second chimeric polypeptide furthercomprises one or more additional target-binding domain(s).
 10. Themulti-chain chimeric polypeptide of claim 1, wherein the soluble tissuefactor domain is a soluble human tissue factor domain.
 11. Themulti-chain chimeric polypeptide of claim 1, wherein the soluble humantissue factor domain comprises a sequence that is at least 95% identicalto SEQ ID NO:
 1. 12. The multi-chain chimeric polypeptide of claim 1,wherein the pair of affinity domains is a sushi domain from an alphachain of human IL-15 receptor (IL- 15Rα) and a soluble human IL-15. 13.The multi-chain chimeric polypeptide of claim 1, wherein the firsttarget-binding domain comprises a sequence that is at least 95%identical to SEQ ID NO:
 85. 14. The multi-chain chimeric polypeptide ofclaim 1, wherein the first target-binding domain comprises a sequencethat is at least 99% identical to SEQ ID NO:
 85. 15. The multi-chainchimeric polypeptide of claim 1, wherein the first target-binding domaincomprises a sequence of SEQ ID NO:
 85. 16. The multi-chain chimericpolypeptide of claim 1, wherein: the first chimeric polypeptidecomprises a sequence of SEQ ID NO: 133; and the second chimericpolypeptide comprises a sequence of SEQ ID NO:
 177. 17. The multi-chainchimeric polypeptide of claim 16, wherein: the first chimericpolypeptide comprises a sequence of SEQ ID NO: 135; and the secondchimeric polypeptide comprises a sequence of SEQ ID NO:
 92. 18. Themulti-chain chimeric polypeptide of claim 1, wherein the secondtarget-binding domain comprises a sequence that is at least 95%identical to SEQ ID NO:
 85. 19. The multi-chain chimeric polypeptide ofclaim 1, wherein the second target-binding domain comprises a sequencethat is at least 99% identical to SEQ ID NO:
 85. 20. The multi-chainchimeric polypeptide of claim 1, wherein the second target-bindingdomain comprises a sequence of SEQ ID NO:
 85. 21. The multi-chainchimeric polypeptide of claim 10, wherein the human soluble tissuefactor domain does not initiate blood coagulation.
 22. A compositioncomprising the multi-chain chimeric polypeptide of claim
 1. 23. Themulti-chain chimeric polypeptide of claim 1, wherein: the first andsecond target-binding domain each comprise a sequence that is at least95% identical to SEQ ID NO: 85; the soluble tissue factor domain is atleast 95% identical to SEQ ID NO: 1; the first domain of the pair ofaffinity domains comprises a sequence that is at least 95% identical toSEQ ID NO: 14; and the second domain of the pair of affinity domainscomprises a sequence that is at least 95% identical to SEQ ID NO: 28.24. The multi-chain chimeric polypeptide of claim 1, wherein the firstdomain of the pair of affinity domains comprises a sequence that is atleast 95% identical to SEQ ID NO: 14, and the second domain of the pairof affinity domains comprises a sequence that is at least 95% identicalto SEQ ID NO:
 28. 25. The multi-chain chimeric polypeptide of claim 1,wherein: the first and second target-binding domain each comprise asequence that is at least 99% identical to SEQ ID NO: 85; the solubletissue factor domain is at least 99% identical to SEQ ID NO: 1; thefirst domain of the pair of affinity domains comprises a sequence thatis at least 99% identical to SEQ ID NO: 14; and the second domain of thepair of affinity domains comprises a sequence that is at least 99%identical to SEQ ID NO:
 28. 26. The multi-chain chimeric polypeptide ofclaim 1, wherein the first domain of the pair of affinity domainscomprises a sequence that is at least 99% identical to SEQ ID NO: 14,and the second domain of the pair of affinity domains comprises asequence that is at least 99% identical to SEQ ID NO:
 28. 27. Themulti-chain chimeric polypeptide of claim 1, wherein the first chimericpolypeptide comprises a sequence that is at least 90% identical to SEQID NO: 133, and the second chimeric polypeptide comprises a sequencethat is at least 90% identical to SEQ ID NO:
 177. 28. The multi-chainchimeric polypeptide of claim 1, wherein the first chimeric polypeptidecomprises a sequence that is at least 95% identical to SEQ ID NO: 133,and the second chimeric polypeptide comprises a sequence that is atleast 95% identical to SEQ ID NO:
 177. 29. The multi-chain chimericpolypeptide of claim 1, wherein the first chimeric polypeptide comprisesa sequence that is at least 99% identical to SEQ ID NO: 133, and thesecond chimeric polypeptide comprises a sequence that is at least 99%identical to SEQ ID NO: 177.